Universiteit van Amsterdam
Bachelor Thesis Scheikunde
The use of Phos-tag to monitor the phosphorylation cascade of the proteins
belonging to the Sln1p two-component pathway and its fusion
with diverse LOV domains
door
Nikki in ‘t Ven
21 januari 2013
Onderzoeksinstituut
Verantwoordelijk docent
Swammerdam Institute for Life Sciences
dhr. prof.dr. K.J Hellingwerf
Onderzoeksgroep
Begeleider
Molecular Microbial Physiology
Aleksandra Bury
Table of content
1
2
3
4
5
6
7
8
Content
Dutch popular abstract
Abstract
Introduction
Materials and methods
Results and discussion
Conclusions and future outlook
References
Appendices
Page number
2
3
4
8
10
19
20
21
1. Dutch popular Abstract
Het doel van dit project is om specifieke en gefuseerde eiwitten van S. cerevisiae, de meest
gebruikte gistsoort voor bijvoorbeeld het bakken van brood of het bereiden van bier, te
klonen produceren en zuiveren. Deze specifieke eiwitten zijn onderdeel van een
signaleringsroute voor stress. Wanneer er een stressvolle situatie voor de cel optreedt, reageert
hij door middel van een fosforylatie cascade. Het eiwit Sln1p fosforyleert Ypd1, een
transporteiwit, en die fosforyleert op zijn beurt de response regulatoren Skn7 en Ssk1, wat een
tegenreactie op de stress in werking zet (zie figuur 2). Om een beter begrip te krijgen hoe dit
soort processen werken is er een fusie gemaakt tussen Sln1p en een LOV-domein van een
ander eiwit. Een LOV-domein reageert op lichtstress, dus op deze manier zou het mogelijk
moeten zijn om een fosforylatie cascade in gang te zetten door middel van licht (of juist de
afwezigheid van licht) in plaats van andere stress. Om te zien of deze fusie-eiwitten werken
wordt die fosforylatie cascade gevolgd door middel van een Phos-tag gel. Gefosforyleerde
eiwitten bewegen langzamer door deze gel dan hun niet-gefosforyleerde tegenhangers omdat
ze interacties aangaan met de Phos-tag. Zo zou je kunnen zien of een eiwit gefosforyleerd kan
worden en of hij die fosfaat ook weer kan doorgeven. Het is met wat moeite gelukt om een
aantal eiwitten te klonen en er een beetje van te produceren, in ieder geval genoeg voor
fosforylatie experimenten. Deze hebben helaas (nog) niet kunnen aantonen dat de fusieeiwitten werken. Dit kan er aan liggen doordat tijdens het fuseren de werking van de eiwitten
verloren is gegaan, maar er waren ook behoorlijk wat problemen met de Phos-tag gel zelf,
waardoor dit nog niet met zekerheid te zeggen is.
2
2. Abstract
The main goal of this project is to optimize the assay for detection of the phosphorylated and
non-phosphorylated form of the proteins of the Sln1p pathway of S. cerevisiae through a
phosphorylation assay. This is to determine if the proteins belonging to the Sln1p twocomponent pathway and its fusion with diverse LOV domains are able to phosphorylate the
two downstream response regulators, Ssk1 and Skn7. For this it is necessary to clone,
overproduce and purify some of the proteins belonging to the Sln1p two-component pathway
and its fusion with diverse LOV domains.
To overproduce these fusion proteins they first have to be cloned from the pQE30 vector to
the pET28b vector and then transformed into E. coli Bl21 cells. These cloned fusion
constructs and the other available proteins of the Sln1p pathway are then overproduced by
inducing them by adding IPTG and purifying them using a 5-ml HisTrap HP Ni-column with
an imidazole gradient. After overnight dialysis they were analysed by Phos-tag SDS-PAGE to
see if a phosphorylation cascade takes place from the fusion proteins C1,C2 and C2A to the
downstream response regulators Skn7 and Ssk1. As a positive control for these assays and for
a protocol of overproduction the ArcA protein was used.
It is quite difficult to clone and transform the fusion constructs C1, C2 and C2A, but by
repeating it often enough it eventually works. The overproduction of the fusion proteins and
the other proteins of the Sln1p pathway did not result in a high yield of protein, but when
repeated often enough there will be enough protein to concentrate them and use them for a
phosphorylation assay. The best conditions turned out to be the same as for the
overproduction of ArcA: an IPTG-concentration of 1 mM, overnight induction at room
temperature and sonication as a lysing method. Until now the Phos-Tag SDS PAGE did not
show a phosphorylation cascade of the Sln1p pathway or of any individual protein of the
pathway. This could be because of the problems with the Phos-tag gel, leaking of the castingsystem and/or low concentrations of protein, or because the proteins are not active, what
would mean that the fusion proteins are not able to perform their function in the stress
response.
3
3. Introduction
The main goal of this project is to optimize the assay for detection of the phosphorylated and
non-phosphorylated form of the proteins of the Sln1p pathway of S. cerevisiae through a
phosphorylation assay. This is to determine if the proteins belonging to the Sln1p twocomponent pathway and its fusion with diverse LOV domains are able to phosphorylate the
two downstream response regulators, Ssk1 and Skn7. For this it is necessary to clone,
overproduce and purify some of the proteins belonging to the Sln1p two-component pathway
and its fusion with diverse LOV domains.
This bachelor project is a small part of a larger project to research the spatial design of
biochemical regulation networks. A part of this research is to investigate photoactivable
protein networks in bakers yeast. One of these networks is the Sln1p two-component pathway
of Saccharomyces cerevisiae. S. cerevisiae Sln1p is a hybrid protein, containing both kinase,
histidine kinase and aspartate receiver domains, which is associable with the plasma
membrane and plays a prominent role in the response of several eukaryotic organisms to the
extracellular environment. Upon osmotic stress the Sln1 histidine kinase de-phosphorylates
the H567 which starts a phosphorylation cascade to first the phosphoaccepting aspartate
D1144 within the receiver domain of Sln1.This is followed by a phosphotransfer step to
histidine H64 in the phosphotransfer protein Ypd1. The last step is between the H64 of Ypd1
and the phosphoaccepting aspartate D554 in the receiver domain of the cytoplasmic response
regulator, Ssk1 and between H64 of Ypd1 and aspartate D427 in the receiver domain of the
nuclear response regulator, Skn7 (figure 1).
Figure 1: phosphorylation cascade of the Sln1p two-component pathway1
The non-phosphorylated form of the cytoplasmic response regulator Ssk1 activates the Hog1
MAP kinase pathway and makes sure that the cell responds upon osmotic stress and the
phosphorylated form of the nuclear response regulator Skn7 is a transcription factor that
makes sure that the cell responds upon wall stress (figure 2)1,2.
4
Figure 2: The Sln1p two-component pathway of S.
cerevisiae1
A promising way to look at the role of compartmentalisation and diffusion in signal
transmission is the use of optogenetics. This is a technique that combines the use of light and
genetically encoded light-sensitive proteins to control the behaviour of living cells and
organisms3,4. An example of a light sensitive protein is YtvA from B. Subtilis. It is a
photoreceptor that consists of an N-terminal LOV(light, oxygen, and voltage)-domain and a
C-terminal STAS(sulphate transporter and anti-sigma factor)-domain, and upon blue light
absorption it elicits the general stress response5. A fusion of the proteins YtvA and Sln1p
could result in a light dependent histidine kinase consisting of a LOV domain from YtvA and
a histidine kinase domain from Sln1p that allows to trigger the phosphorylation reaction by
light instead of osmotic stress6 (figure 3). If the fusion protein elicits a stress response upon
light absorption or on the absence of light is not clear yet.
Figure 3: Sln1p histidine kinase fused with a LOV domain from YtvA with receiver domain
(upper) and without receiver domain (lower)
A route to determine if these fusion proteins are active and able to trigger the phosphorylation
cascade is the use of Phos-tag SDS-PAGE. Phos-tag is a binuclear Mn2+ complex with an
acrylamide-pendant Phos-tag ligand. A phosphorylated protein binds to the two Mn2+ ions at
the phosphate binding site, which results in separation of the phosphoproteins from their
nonphosphorylated counterparts. The phosphorylated proteins migrate slower in SDS-PAGE
than do the corresponding nonphosphorylated molecules, because the phospho group interacts
with the Mn2+ Phos-tag ligand in the gel (figure 4)7,8,9.
5
The advantages of using Phos-tag SDS-PAGE for the mobility shift detection of
9
phosphorylated
Figure 4: Phosphate-affinity
proteins are
Mn:2+-Phos-tag SDS-PAGE for the mobility-shift detection of phosphoproteins9
- Radioactive and chemical labels are avoided.
- Phosphoprotein isotypes can be detected as multiple migration bands in the same lane.
- The procedure is almost the same as that for the general SDS-PAGE.
- The binding specificity of Phos-tag is independent of amino acid and sequence context.
- Downstream procedures such as Western blot analysis and MS analysis are applicable.
- Phos-tagTM AAL-107 dissolved in distilled water is stable for at least 3 months.
- The time-course ratio of phosphorylated and non-phosphorylated proteins can be
determined.
- Separation of phosphoprotein isotypes having the same number of phosphate groups is
possible.
At the start of the project there were two fusion constructs available, C1 LOV(YtvA)Sln1p
linker from YtvA and C2 LOV(YtvA)Sln1p linker from Sln1p. All the constructs available at
the start of this project are listed in table 1.
Table 1: Constructs available at the start of this project
1
2
3
4
5
6
7
8
Construct
C1 LOV(YtvA)Sln1p linker from YtvA
C2 LOV(YtvA)Sln1p linker from Sln1p
C2A LOV(YtvA)Sln1pHK
Skn7
Skn7Rec
Ssk1
Ssk1Rec
Ypd1
Size (bp)
2401
2442
393+1281=1675
1869
789
2138
951
504
pQE30
+
+
pET28b
+
+
+
+
Before the start of the project there were already attempts made to overproduce C1 and C2 in
pQE30, but they weren’t successful, so the first step is to clone them from pQE30 to pET28b.
This because the pET System is the most powerful system yet developed for the cloning and
expression of recombinant proteins in E. coli10. C2A LOV(YtvA)Sln1pHK, a construct that is
the same as C2 but then without the receiver domain of Sln1p (figure 3), is also cloned to
pET28b by using a different RV primer (appendix 1) with the PCR-reaction of C2 in pQE30.
The primers used in these PCR-reactions also encode the restriction sites necessary to clone
them into pET28b, because pQE30 and pET28b don’t have complementary restriction sites in
their Multiple Cloning Site (figure 5).
Figure 5: The vectors pQE30 and pET28b with their Multiple Cloning Sites
6
Furthermore, C1 is restricted with SalI instead of XhoI, one of the enzymes that is used to
digest pET28b, because it has another restriction site within the sequence what would lead to
cleavage. The restriction with SalI should still work, because SalI and XhoI produce
compatible sticky ends.
These cloned fusion constructs and the other available proteins will then be overproduced in
E. coli Bl21 cells and analysed by Phos-tag SDS-PAGE to see if a phosphorylation cascade
takes place from the fusion proteins C1, C2 and C2A to the downstream response regulators
Skn7 and Ssk1. As a positive control for these assays and for a protocol of overproduction the
ArcA protein will be used11.
7
4. Materials and methods
4.1 Cloning and transformation
For the cloning of the constructs C1, C2 and C2A (see table 1) from pQE30 to pET28b, 4
PCR reactions were carried out with pQE30(LOV(YtvA)Sln1p, linker from YtvA) and
pQE30(LOV(YtvA)Sln1p,linker from Sln1p) as a DNA template following protocol
(appendix 2). Reaction 1 contained pQE30(LOV(YtvA)Sln1p,linker from YtvA) as a template
DNA and the primers pET28bYtvAFW and pQE30Sln1pRV. Reaction 2 contained
pQE30(LOV(YtvA)Sln1p,linker from Sln1p) as a template DNA and the primers
pET28bYtvAFW and pET28bSln1pRV. Reaction 3 contained
pQE30(LOV(YtvA)Sln1p,linker from Sln1p) as a template DNA and the primers
pET28bYtvAFW and pET28bSln1pHKRV and reaction 4 contained only the primers
pET28bYtvAFW and pQE30Sln1pRV as a control (for sequences of the primers see appendix
1). The products were analysed on a 1% agarose gel, purified with a MSB Spin PCRapace
500 kit from Invitek and the concentration was determined by Nanodrop.
20 ml cultures of E .coli BL21 which contained pET28b were grown in 200 ml conical flasks
with vigorous shaking (200 rpm) on a rotary shaker at 37°C in LB medium (10 g·liter−1
tryptone, 5 g·liter−1 yeast extract, 5 g·liter−1NaCl). Kanamycin was routinely included at a
final concentration of 50 μg·ml−1. After overnight growth the plasmid was purified with a kit
column plasmid miniprep classic from QIAGEN.
Next, the purified PCR products and the plasmid were digested/restricted using FastDigest
enzymes by Fermentes. 0.2μg of the plasmid DNA from reaction 1 was restricted with 1μl
NdeI and 1μl SalI, 0.2μg of the plasmid DNA from reaction 2 was restricted with 1μl NdeI
and 1μl XhoI, 0.2μg of the plasmid DNA from reaction 3 was restricted with 1μl NdeI and 1μl
SalI and 1μg of the purified plasmid was restricted with 1μl NdeI and 1μl SalI. Each reaction
mixture contained 2μl FastDigest buffer and the volume was made up to 20μl with H2O. After
1h of vigorous shaking (200 rpm) on a rotary shaker at 37°C, 2μl of FastAP was added to
reaction mixture 4 and after 75min shaking they were all purified with a MSB Spin PCRapace
kit from Invitek, analysed on a 1% agarose gel and the concentration was determined by
Nanodrop.
Then ligation mixtures were prepared, each containing 1μl T4 ligase, 1μl ligase buffer and
different ratios of vector(purified restriction mixture 4) : insert(purified restriction mixtures 1
to 3). To 1 mixture H2O was added instead of an insert as a control.
For the transformation of the ligation mixtures into electro competent E. coli Bl21 cells
(prepared following protocol, see appendix 3) the constructs were first transformed into
chemically competent E. coli XL1-Blue cells. The ligation mixtures were added to the
competent cells in different ratios and transformed following protocol (appendix 4). They
were plated out on LB plates with Kanamycin at a final concentration of 50 μg·ml−1. After
overnight incubation at 37°C the transformations were checked with colony PCR (appendix 5)
and an 1 % agarose gel.
When the transformation succeeded, a glycerol stock was made and the constructs were send
for sequencing. The glycerol stock contained 250μl 60% glycerol and 750μl of overnight 20
ml cultures of the transformed cells containing the construct, that were grown in 200 ml
conical flasks with vigorous shaking (200 rpm) on a rotary shaker at 37°C in LB medium with
8
Kanamycin at a final concentration of 50 μg·ml−1. For the sequencing, these overnight
cultures were purified using a QIAGEN QIAprep Spin Miniprep, the concentrations were
determined by Nanodrop and per construct 2 reaction mixtures were prepared and send for
sequencing. One mixture contained 0.5μl of the appropriate FW primer, 1-1.5μg of the
purified construct and the volume was made up to 6.5μl with H2O. The other mixture
contained the corresponding RV primer instead of the FW primer(for primers see appendix 1).
The same procedure was followed for the cloning of the constructs Ssk1Rec and Skn7 from
pET28b to pQE30.
The FW and RV primers for Ssk1Rec were pET28sskRecFW and pETssk1RV and for Skn7,
pET28sskn7FW and pET28sskn7FW (for sequences see appendix 1)
20 ml cultures of pQE30 were grown in 200 ml conical flasks with vigorous shaking (200
rpm) on a rotary shaker at 37°C in LB medium. Ampicillin was routinely included at a final
concentration of 100 μg·ml−1.
Ssk1Rec and pQE30 were restricted with BamHI and HindIII and Skn7 and pQE30 with SphI
and HindIII.
The constructs were first transformed into chemically competent E. coli XL1-Blue cells and
next in electro competent E. coli M15 cells.
The E. coli XL1-Blue cells were plated out on LB plates with Kanamycin at a final
concentration of 50 μg·ml−1 and the E. coli M15 cells were plated out on LB plates with
Kanamycin at a final concentration of 25 μg·ml−1.and Ampicillin at a final concentration of
100 μg·ml−1.
4.2 Overproduction of some of the proteins belonging to the Sln1p twocomponent pathway and its fusion with diverse LOV domains
For overproduction of the His6-tagged Sln1p proteins, 20 ml cultures of the transformed E.
coli strain BL21 were grown in 200 ml conical flasks with vigorous shaking (200 rpm) on a
rotary shaker at 37°C in LB medium (10 g·liter−1 tryptone, 5 g·liter−1 yeast extract, 5
g·liter−1NaCl). Kanamycin was routinely included at a final concentration of 50 μg·ml−1 for
plasmid maintenance. After overnight growth the 20 ml cultures were poured in 1-liter fresh
LB-medium with Kanamycin at a final concentration of 50 μg·ml−1in 5-liter conical flasks
and grown with vigorous shaking (200 rpm) on a rotary shaker at 37°C. When the culture
attained an optical density at 600 nm (OD600) of approximately 0.6, induction of protein
production was initiated by adding isopropyl-β-d-thiogalactopyranoside (IPTG). After
growth, the cells were harvested by centrifugation, and the cell pellet could be stored at
−20°C until use. All subsequent steps were performed at 4°C and on ice.
Next, the cell pellet was resuspended in 40 ml of a lysis buffer (10 mM NaCl, 15% glycerol,
50 mM Tris-HCl; pH 8), containing 1.3 mg·ml−1 lysozyme, 30 μg·ml−1 DNase and RNase.
After 30 min of incubation at room temperature, the cells were lysed. And immediately
following cell lysis, a protease inhibition cocktail was added. The resulting cell lysate was
then clarified by centrifugation at 15,000 rpm for 30 min.
The cell lysate was then filtered and applied to a 5-ml HisTrap HP Ni-column (GE healthcare)
equilibrated with buffer A (500 mM NaCl, 50 mM Tris-HCl; pH 8, 20 mM imidazole). After
the column was washed with buffer A, the protein was injected in to the column and eluted
with buffer B (500 mM NaCl, 50 mM Tris-HCl; pH 8, 500 mM imidazole). The fraction with
eluted protein was collected in five 2-ml fractions. These fractions were immediately dialyzed
against 20 mM Tris-HCl (pH 8).
After dialysis the fractions were analysed by fast SDS PAGE and the concentration was
determined by a BSA Biorad assay.
9
Al the Skn7Rec fractions were put together, ass well as the C2A fractions, and they were
concentrated with a Spin-X UF concentrator 10kd from corning at 4000 rpm for 20 min at
4˚C. After concentrating, the fractions were analysed by fast SDS PAGE. Next, the C2A
fractions were concentrated with a Spin-X UF concentrator 50kd from corning at 4000 rpm
for 15 min at 4˚C and again analysed by fast SDS PAGE.
4.3 Phosphorylation assay
For the phosphorylation assay of the Sln1p proteins, the SDS PAGE Bio-rad system and
Acrylamide-pendent Phos-tag AAL-107 were used.
To phosphorylate the proteins, they were incubated for at least 1.5 h in a phosphorylation
buffer (table 2) with different concentrations of acetyl phosphate and different ratios H2O :
protein, depending on the protein concentration Before they were loaded on the gel they were
mixed 2:1 with loading buffer which containing SDS, mercaptoethanol, glycerol and
bromophenol blue. The unphosphorylated proteins were also diluted with the same loading
buffer, with different dilution factors depending on the protein concentration.
Table 2: The components of the phosphorylation buffer and the stacking and resolving gel solutions
Phosphorylation buffer:
30 mM HEPES pH 7.5
10 mM MgCl2
10% glycerol
10, 50 mM acetyl phosphate
Protein
H2O
Resolving gel solution:
20% acrylamide/bis solution
Lower gel buffer pH 8.8 (Trizma, SDS)
5 mM PhosTag
10 mM MnCl2
Distilled water
TEMED
10% APS
At least 1.5 h incubation in 30ºC 1h to 3h
Stacking gel solution
20% acrylamide/bis
solution
Lower gel buffer pH 6.5
(Trizma, SDS)
Distilled water
TEMED
10% APS
The required solutions, buffers and the loading of the gels were al done following protocol
(appendix 6) The electrophoresis ran 1.5 h to 3 h at 25 mA current per gel.
The gels were washed with demi-water, boiled with Coomassie Brillant Blue staining in a
microwave, and left to cool for 20 min with slow agitation. Next, they were washed with
demi-water, boiled with de-staining solution and left to cool for 20 min with slow agitation.
After the boiling steps the gels were washed with demi-water and left in demi-water overnight
with slow agitation.
5 Results and Discussion
The main goal of this project is to optimize the assay for detection of the phosphorylated and
non-phosphorylated form of the proteins in the Sln1p pathway through a phosphorylation
assay. This is to see if the proteins belonging to the Sln1p two-component pathway and its
fusion with diverse LOV domains are able to phosphorylate the two downstream response
regulators. For this it is necessary to clone, overproduce and purify some of the proteins
belonging to the Sln1p two-component pathway and its fusion with diverse LOV domains. As
a positive control for the overproduction and assays the ArcA protein was used.
10
5.1 Cloning and transformation of C1, C2 and C2A
The first step is to clone the LOV Sln1p fusion proteins C1, C2 and C2A from pQE30 to
pET28b.
Table 3: Constructs available at the start of this project
Construct
C1 LOV(YtvA)Sln1p linker from YtvA
C2 LOV(YtvA)Sln1p linker from Sln1p
C2A LOV(YtvA)Sln1pHK
Skn7
Skn7Rec
Ssk1
Ssk1Rec
Ypd1
1
2
3
4
5
6
7
8
a
Size (bp)
2401
2442
393+1281=1675
1869
789
2138
951
504
pQE30
+
+
pET28b
+
+
+
+
b
PCR reaction:
2000bp
1
2
3
4
1.
2.
3.
4.
5.
5
Restriction:
C1
C2
C2A
H2O
Ladder
2000bp
1
2
3
4
5
6
1.
2.
3.
4.
5.
6.
C1, NdeI, SalI
C2, NdeI, XhoI
C2A, NdeI, XhoI
pET28b, NdeI, XhoI
Purified pET28b
Ladder
Figure 6: (a) C1, C2 and C2A after PCR reaction with the right sizes (for expected sizes see table 3) (b) C1, C2, C2A and pET28b after
restriction. C1 and C2 have different sizes now, because they are digested with different restriction enzymes
After the PCR reactions and the restrictions succeeded (figure 6), ligation mixtures were
prepared with different ratios of vector(restricted pET28b) : insert(restricted C1,C2,C2A) and
transformed into electrocompetent (EC) cells or chemically competent (CC) cells with
different ratios of ligation mixtures : competent cells (table 4).
Table 4: Different attempts to transform C1, C2, C2a, Skn7 and Ssk1Rec into various electrocompetent (EC) or chemically
competent (CC) E. coli cells
Constructs
1
2
3
C1,C2,C2A
C1,C2,C2A
C1,C2,C2A
4
5
6
7
8
9
10
11
12
13
C1,C2,C2A
C1,C2
C1,C2
C1,C2
XL1-Blue C2A
Skn7
Skn7
Skn7
Ssk1Rec
XL1-Blue Ssk1Rec
Ligation
Vector:Insert
1μl:3μl
1μl:3μl
Exactly calculated from
concentration restriction
1μl:3μl
1μl:7μl
1μl:7μl
1μl:3μl
1μl:7μl
1μl:7μl
1μl:3μl
1μl:7μl
-
Cells
(E. coli)
EC BL21
EC BL21
CC XL1-Blue
Transformation
Ligation:Cells
1μl:200μl
1μl:200μl
2μl:20μl
Transformed
CC XL1-Blue
CC XL1-Blue
CC XL1-Blue
CC XL1-Blue
EC BL21
CC XL1-Blue
CC XL1-Blue
CC XL1-Blue
CC XL1-Blue
EC pQE30
2μl:20μl
4μl:30μl
4μl:20μl
3μl:50μl
0.5μl:200μl
3μl:50μl
3μl:50μl
3μl:50μl
3μl:50μl
0.5μl:200μl
C2A
C2A
Skn7
Ssk1Rec
Ssk1Rec
-
11
The first two attempts were with a standard ratio of vector:insert and directly transformed to
E. coli BL21. There were a few colony’s, between 1 to 10 per construct, but after colony PCR
there were no products. The next step to transform the constructs was to try to transform them
first into commercially available chemically competent E. coli XL1-Blue cells. These cells are
endonuclease (endA) deficient, which greatly improves the quality of miniprep DNA, and are
recombination (recA) deficient, improving insert stability. It also has a hsdR mutation which
prevents the cleavage of cloned DNA by the EcoK endonuclease system12. The second try
succeeded for the construct C2A, with a standard ligation ratio of 1:3 and standard
transformation ratio of 2:20 (figure 7a). It was send for sequencing with both the forward
primer JBS60/T7_FW and the reverse primer JBS61/T7_RV, because C2A is to large for one
sequence reaction. Normally one sequence reaction can sequence about 1000bp correctly and
C2A has 1675bp. The first reaction with the forward primer managed to sequence up to about
950bp and the backward primer from about 850bp. Because the two sequences overlap, the
sequencing confirmed that it is the right construct and it also confirmed that C2A transformed
in E. coli XL1-blue has no mutations (appendix 7).
The other two constructs, C1 and C2, had again few colony’s, between 1 to 10 per construct,
and after colony PCR no products. The transformation of C1 and C2 was repeated several
more times with different ligation and transformation ratios, but that did not work as well.
Because of time constraints the attempts stopped there, but someone else kept on trying and
eventually made it work, with standard ligation and transformation ratios.
Next, C2A was transformed from E. coli XL1-Blue to E. coli Bl21 and that succeeded in one
try (figure 7b).
The reason why these constructs were difficult to clone could be that there quite large, there
are more sites for mutation and cleavage. Earlier cloning and transformation into other vectors
and cells of these and other comparable constructs also displayed difficulties.
a
Colony PCR:
2000bp
1
2
3
4
5
6
1–4. Colony’s from
E.coli XL1-Blue
5. Ladder
b
Colony PCR:
1. Ladder
2-9. Colony’s from
E. coli BL21
2000bp
1
2
3 4 5 6
7 8 9
Figure 7: (a) Product of the colony PCR on the E .coli XL1-Blue transformed with pET28bC2A (expected size 1675bp)
(b) Product of the colony PCR on the E. coli BL21 transformed with pET28bC2A (expected size 1675bp)
5.2 Overproduction of some of the proteins belonging to the Sln1p twocomponent pathway and its fusion with diverse LOV domains
For the overproduction of the proteins belonging to the Sln1p two-component pathway, the
procedure of the overproduction of the protein ArcA was used. The conditions for the
overproduction of Arc are an IPTG concentration of 1mM, overnight overexpression at room
temperature(RT) and sonication as a lysing method. Therefore for the first attempt of
overproducing one of the downstream response regulators, Skn7Rec, the same conditions
were used.
12
This did not have the desired results, a concentration of 14μM Skn7Rec compared to a
concentration of 25μM ArcA, calculated with a BSA BioRad assay.
The next step was to differ the conditions for the overproduction, to see if there is any
improvement possible and eventually determine what conditions are the best. The IPTG
concentration was changed to 0.5 mM, the time and temperature of IPTG cultivation was
altered and the cells were lysed in a different way, with a French press instead of sonication.
This technique results in more uniform and complete disruption than is possible with
mechanical or ultrasonic methods. None of these changes or combination of changes resulted
in a higher yield then the first attempt (table 4).
Table 5: Different conditions for the overproduction of the proteins Skn7Rec, Skn7, C2A and Ssk1Rec
Attempts
Protein
IPTG
concentration
Temperature
Time of IPTG
overexpression
Lysing
method
Concentration
(BSA BioRad assay)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
ArcA
Skn7Rec
Skn7Rec
Skn7Rec
Skn7Rec
Skn7Rec
Skn7Rec
1 mM
1 mM
1 mM
0.5 mM
1 mM
0.5 mM
1 mM
RT
RT
37˚C
RT
30˚C
30˚C
RT
o/n
o/n
3h
3h
o/n
o/n
o/n
Skn7Rec
0.5 mM
RT
o/n
Skn7
Skn7
1 mM
0.5L 1mM
0.5L 20 μM
1 mM
0.5L 1 mM
0.5L 20 μM
1 mM
RT
RT
RT
RT
RT
RT
RT
o/n
o/n
o/n
o/n
o/n
o/n
o/n
Sonication
Sonication
French press
French press
Sonication
Sonication
Sonication
French press
Sonication
French press
Sonication
Sonication
Sonication
Sonication
Sonication
Sonication
Sonication
25 μM
14 μM
3 μM
5 μM
6 μM
7 μM
-
C2A
C2A
Ssk1Rec
For attempts 7-10 (table 4) the concentration was not determined with a BSA BioRad assay,
because it was already clear from the height of the protein peaks from the FPLC graph that
there was no improvement (see appendix 8 and compare attempt 1, 2, 3 and 7).
When the procedure is repeated often enough, there is enough protein produced to use in a
phosphorylation assay. That is why, after 10 attempts of overproducing Skn7Rec, the switch
was made to overproduce other proteins belonging to the Sln1p two-component pathway:
full length Skn7, Ssk1Rec and the cloned C2A. With these attempts the IPTG concentration
was even lowered to 20μM, but also with these attempts there was no improvement, it got
even worse (see appendix 8).
To see where the problem lies, not only the produced protein was analysed with Fast SDSPAGE, but also the membrane pellet (at the correct dilution factor), flow through, (the
fraction that comes out during the loading of the sample before the elution starts with buffer
B) and cell-free extract were loaded on the gel (figure 8)
13
Fast SDS-PAGE
Skn7Rec
a
Fast SDS-PAGE
C2A
b
35kDa
1
2
3
4
1. Skn7Rec
2. Flow through
3. Cell free extract
4. Pellet
5. Page ruler plus
prestained
5
55kDa
15kDa
1
2
3
4
5
1. Pellet
2. Cell free extract
3. Flow through
4. C2A
5. Page ruler plus
prestained
Figure 8: (a) Skn7Rec with the right size (expected size about 29kDa) (b) C2A seems to be degraded and split into its LOVdomain and HK-domain (expected sizes LOV about 14.5kDa and HK about 47kDa)
Both with Skn7Rec and C2A there is still some protein left in the pellet, as well as in the flow
trough and the cell free extract. In these fractions, C2A also seems to be degraded and split
into its LOV-domain and HK-domain. Although there is some protein in the other fractions, it
is not that much that it explains the low amount of overproduction of the protein.
On the lane where the overproduced C2A should be is nothing to see, but there was a small
peak on the FPLC graph from the Ni-column purification (appendix 8), so there is some
overproduction of C2A.
The next step was to concentrate the combined Skn7Rec and C2A fractions to concentrate
them enough so that they could be used for the phosphorylation assay.
Fast SDS-PAGE C2A and
Skn7Rec
55kDa
35kDa
1
2
3
4
5
1. Cell free extract C2A
2. Conc. C2A
3. Cell free extract Skn7Rec
4. Conc. Skn7Rec
5. Page ruler plus prestained
Figure 9: Concentrated C2A and Skn7Rec with the right sizes (expected 61.5kDa and 29kDa)
Skn7Rec is pure and C2A has still some impurities, but it seems that it is not degraded (figure
9). The concentration of the concentrated Skn7Rec is 20μM, determined with a BSA BioRad
assay. To get rid of the impurities C2A was again concentrated, but this time with a 50 kDa
membrane instead of 10 kDa. Everything smaller than 50 kDa should go through the
membrane, but not C2A, that has a size of about 60 kDa. After the extra concentration there
was less then 1 ml left and it had a yellow colour, a sign that the LOV-domain is present, but
there was not enough left to determine the concentration.
C2A
55kDa
1
2
1. Extra conc. C2A
2. Conc. C2A
3. Page ruler plus prestained
3
Figure 10: Extra concentrated C2A with still some impurities
present (expected size of C2A is 61.5kDa)
14
The Fast SDS-PAGE shows that there are still impurities present, not everything went through
the membrane (figure 10). A way to avoid this could be to spin the filter for a few minutes,
invert it so that the membrane does not get clogged up, and repeat this a few times.
Because of the problems with overproduction of the downstream response regulators Skn7
and Ssk1 in E. coli Bl21, they were cloned from pET28b to pQE30 and transformed into
commercially available E. coli M15, to see if these cells will be better in overproducing the
proteins.
After the PCR reactions and the restrictions succeeded (figure 11), ligation mixtures were
prepared with different ratio’s of vector(restricted pQE30) : insert(restricted Skn7,Ssk1) and
transformed into competent E. coli cells with different ratios of ligation mixtures : competent
cells (table 4).
a
b
Restriction:
PCR reaction:
2000bp
1.
2.
3.
4.
Skn7
Ssk1
H2O
Ladder
2000bp
1
2
3
4
1.
2.
3.
4.
5.
Skn7, BamHI, HindIII
Ssk1, SphI, HindIII
pQE30, BamHI, HindIII
pQE30, SphI, HindIII
Ladder
5
Figure 11: (a) Skn7 and Ssk1 (upper bond) after PCR reaction with the right sizes (expected sizes 1869bp and 2138bp)
(b) Skn7, Ssk1 and pQE30 after restriction with the right sizes (expected sizes 1869bp and 2138bp)
3
The transformation of Ssk1 into E. coli XL1-Blue succeeded right away (figure 12a) and this
was send for sequencing with both the forward primer JcHpQE30_FW and the reverse primer
JcHpQE30_RV, because Ssk1 has a size of 2138bp. The first reaction with the forward primer
managed to sequence up to about 950bp and the backward primer from about 1000bp. The
two sequences do not overlap, but the part that is sequenced is the right construct and it has no
mutations but one at the end (appendix 9).
The transformation of E. coli M15 with pQE30Ssk1 also succeeded at the first attempt (figure
12b).
4
a
b
Colony PCR
2000bp
1-3. E. coli
XL1-Blue
Ssk1Rec
4. Ladder
Colony PCR
2000bp
1-4. E. coli
M15
Ssk1Rec
5. Ladder
1
2
3
4
1
2
3
4
5
5Figure 12: (a) Product of the colony PCR on the E. coli XL1-Blue transformed with pQE30Ssk1(expected size 2138bp)
(b) Product of the colony PCR on the E. coli M15 transformed with pQE30Ssk1 (expected size 2138bp)
Three attempts with different ratios of ligation and transformation were necessary to
transform Skn7 into E. coli XL1-Blue (figure 13 and table 4).
15
2000bp
Colony PCR
1-4. E. coli
XL1-Blue
Skn7
5. Ladder
1
2
3
4
5
Figure
13: 2Product
1
3 of the4colony5PCR on the E. coli XL1-Blue
transformed with pQE30Skn7(expected size 1869bp)
Because of time constraints, Skn7 was not transformed any further from E. coli XL1Blue to
E. coli M15 and the proteins were not overproduced yet.
5.3 Phosphorylation assay
The first assay was to determine if Skn7Rec would phosphorylate when incubated with acetyl
phosphate at a final concentration of 10 mM and 50 mM. ArcA was used as a control. The
first attempts did not result in good Phos-tag gels, an example of these gels is figure 14, the
upper gel. The problem here was that the samples ran normally at first, but then all of a
sudden dropped down, which is visible by looking at the ladder. One of the possible problems
mentioned by the supplier of Phos-tag TM is that some prestained protein markers could
interact with the Phos-tag gel resulting in broad and/or distorted bands. Therefore the next
step was to avoid using the prestained ladder, but instead using three proteins with known
size, see figure 14 the lower gel. This did not solve the problem, the samples did not ran at all.
Upper gel:
1. Page ruler plus prestained
2. ArcA
3. ArcA 10 mM ac ph
4. Skn7Rec
5. Skn7Rec 10 mM
6. Skn7Rec 50 mM
Lower gel:
1. Albumine
2. Cytochrome C
3. BSA
4. ArcA
5. ArcA 10 mM ac ph
6. Skn7Rec
7. Skn7Rec 10 mM
8. Skn7Rec 50 mM
Figure 14: Phos-tag SDS-PAGE of ArcA and Skn7Rec with and without ladder
Because it was still unclear if Skn7Rec phosphorylates, the Phos-tag gel was made again and
the same samples were loaded, but this time also a regular SDS-PAGE gel was made as a
control (figure 15).
16
1. Page ruler plus prestained
2. Skn7Rec
3. Skn7Rec 10 mM
4. Skn7Rec 50 mM
5. ArcA 10 mM ac ph
6. ArcA
Figure 15: Phos-tag SDS-PAGE and regular SDS-PAGE of Skn7Rec and ArcA
With this attempt the regular SDS-PAGE gel worked and this time also the Phos-tag gel,
although still not without some distortion. In the regular gel the Skn7Rec shows two bands
indicating that it suffered some form of degradation. This is not the case in the Phos-tag gel,
but there is also no sign of phosphorylation of Skn7Rec. Something that is clearly observable
with the control protein ArcA, which shows two bands in lane 5 instead of one in lane 6, with
one band higher indicating that part of the protein is phosphorylated.
1. Page ruler plus prestained
2. C2
3. C2+ATP
4. C2+ATP+Skn7Rec
5. Skn7Rec
6. C1+ATP+Skn7Rec
7. C1+ATP
8. C1
9. ArcA 10 mM ac ph
10. ArcA
Figure 16: Phos-tag SDS-PAGE of C1 and C2 attempting to show a phosphorylation cascade
Because Skn7Rec did not show any phosphorylation until now, it was then incubated with C1
and C2 and ATP, to see if these fusion proteins that are part of the Sln1p pathway could
phosphorylate Skn7Rec. ATP was added because C1 and C2 should have ATPase activity,
and C1, C1 + ATP (same for C2) and ArcA were loaded as a control (figure 16). Although
there are again some distorted bands, the Phos-tag gel worked, indicated by ArcA. C1, C2 and
Skn7Rec do not seem to show any phosphorylation, they all have single bands at about the
same height, but it s still a bit unclear because of the distortion and the low concentration of
C1 and C2.
17
1. Page ruler plus prestained
2. C1
3. C1+ATP+Ypd1
4. C1+ATP+Ypd1+Skn7Rec
5. Ypd1
6. Skn7Rec
7. C2+ATP+Ypd1+Skn7Rec
8. C2+ATP+Ypd1
9. C2
Figure 17: Phos-tag SDS-PAGE of C1 and C2 trying to show a phosphorylation cascade
TEMED and APS and in a different ratio (10μl : 40μl instead of 20μl : 35μl)
The literature says1 that Sln1p can phosphorylate the downstream response regulators Skn7
and Ssk1 straight away without the help of the phosphotransfer protein Ypd1, but to be sure
C1 and C2 were also incubated with this protein (figure 17). Again the proteins do not seem
to show any phosphorylation, they all have single bands at about the same height, but it is still
a bit unclear because of the low concentration of C1 and C2 and the overconcentration of
Ypd1.
There were more attempts made to see if the phosphorylation cascade occurs, for example
with a lower concentration of Ypd1 or with the overproduced and concentrated C2A, but there
were a lot of problems with the Phos-tag gel, resulting in heavily distorted bands from which
no conclusions could be drawn (figure 18). The main problem was leakage of the system
during the setting of the gels. When the set-up of the gel leaks while the Phos-tag gel is setting
the Mn2+ Phos-tag ligands probably do not align properly resulting in a crooked gel and
heavily distorted bands. One of the attempts to try to solve this problem was to poor a thin
layer of 1% agarose, a very fast setting gel, on the bottom of the casting-system to try to seal
it of completely. This worked better, but it still leaked most of the times and even though it
was only a few microliters, it resulted in distorted bands.
Another way was to use TEMED and APS and in a different ratio (10μl : 40μl instead of 20μl
: 35μl) to see if that caused any distortion, and that resulted in one of the nicest gels (figure
17). The only problem with method was that it took the gel at least 3h to set, therefore if the
system leaked just a few microliters the gel was unusable.
1. Page ruler plus prestained
2. C2A
3. C2A+ATP+Ypd1
4. C2A+ATP+Ypd1+Skn7Rec
5. Ypd1
6. Skn7Rec
7. C2A+ATP+Skn7Rec
8. C2A+ATP
Figure 18: Failed Phos-tag SDS-PAGE of C2A
18
6. Conclusions and future outlook
The constructs cloned and transformed during this project are listed in table 5 in green.
Table 6: Constructs available at the end of the project
Construct
Size
C1 LOV(YtvA)Sln1p linker from YtvA
C2 LOV(YtvA)Sln1p linker from Sln1p
C2A LOV(YtvA)Sln1pHK
Skn7
Skn7Rec
Ssk1
Ssk1Rec
Ypd1
2401
2442
393+1281=1675
1869
789
2138
951
504
Cloned
pQE30
pET28b
+
Send for seq.
+
Send for seq.
+
+
+
+
+
+
+
+
Transformed
XL1-Blue Bl21
M15
+
+
+
+
+
It is quite difficult to clone and transform the fusion constructs C1, C2 and C2A, but by
repeating it often enough with the standard ratios of vector:insert 1μl:3μl or 1μl:4μl during
ligation and ligation mixture:cells 3μl:20μl during transformation, it eventually works. The
cloning of Skn7 and Ssk1 was straight forward.
The overproduction of the fusion proteins and the other proteins of the Sln1p pathway did not
result in a high yield of protein, but when repeated often enough there will be enough protein
to concentrate them and use them for a phosphorylation assay. Also if the next step would be
using these proteins for Western blotting , this will be enough, because that only requires less
than 1 microgram of protein. The best conditions turned out to be the same as for the
overproduction of ArcA: an IPTG-concentration of 1 mM, overnight induction at room
temperature and sonication as a lysing method.
It is of course possible to vary the conditions even more to attempt to get a higher amount of
protein, for example inducing at a low temperature of 16-17˚C.
If there would be a lot of protein left in the membrane pellet trapped in inclusion bodies, there
is a method of getting it out of the membrane by using urea13. The problem whit this method
is though, that a part of the protein gets lost. Seeing that there is not a significant amount of
protein in the membrane fraction compared to the protein yield, this would probably be a
waste of time considering how much it could add to the total amount of protein produced.
Inclusion bodies are probably not the problem, there is just not that much protein
overproduced. Another thing to try could be using different cells for the overproduction, like
E. coli M15.
Until now the Phos-Tag SDS PAGE did not show a phosphorylation cascade of the Sln1p
pathway or of any individual protein of the pathway. This could be because of the problems
with the Phos-tag gel or because the proteins are not active, what would mean that the fusion
proteins are not able to perform their function in the stress response. A way to address the
problems with the Phos-tag gel could be to use a casting-system that is not leaking and to use
more concentrated and/or better purified proteins. There is also another way to show
phosphorylation and that is to label the proteins1, but that was one of the advantages of using
Phos-tag SDS-PAGE for the mobility shift detection of phosphorylated proteins, that
radioactive and chemical labels are avoided.
19
7. References
1. Fassler, J. S., and West, A. H. (2010) Genetic and biochemical analysis of the SLN1
pathway in saccharomyces cerevisiae Methods Enzymol. 471, 291-317.
2. Kaserer, A. O., Andi, B., Cook, P. F., and West, A. H. (2010) Kinetic studies of the yeast
his-asp phosphorelay signaling pathway Methods Enzymol. 471, 59-75.
3. Toettcher, J. E., Gong, D., Lim, W. A., and Weiner, O. D. (2011) Light-based feedback for
controlling intracellular signaling dynamics Nat. Methods. 8, 837-839.
4. Editorial (2010) Method of the year Nat. Methods. F.321 vol.8 no.1, januari 2011
5.
Avila-Perez, M., Vreede, J., Tang, Y., Bende, O., Losi, A., Gartner, W., and Hellingwerf,
K. (2009) In vivo mutational analysis of YtvA from bacillus subtilis: Mechanism of light
activation of the general stress response. J. Biol. Chem. 284, 24958-24964.
6.
Moglich, A., Ayers, R. A., and Moffat, K. (2009) Design and signaling mechanism of lightregulated histidine kinases. J. Mol. Biol. 385, 1433-1444.
7. Kinoshita, E., Kinoshita-Kikuta, E., Takiyama, K., and Koike, T. (2006) Phosphate-binding
tag, a new tool to visualize phosphorylated proteins Mol. Cell. Proteomics. 5, 749-757.
8. Barbieri, C. M., and Stock, A. M. (2008) Universally applicable methods for monitoring
response regulator aspartate phosphorylation both in vitro and in vivo using phos-tag-based
reagents Anal. Biochem. 376, 73-82.
9. Kinoshita-kikuta, E. Kinoshita, E. and Koike, T. (2010) Separation and detection of large
phosphoproteins using Phos-tag SDS-PAGE NPG
10. Jiechao, Y. Guangxing, L. Xiaofeng, R. Georg, H. (2007) Select what you need: A
comparative evaluation of the advantages and limitations of frequently used expression
systems for foreign genes J. Biotech. 127, 335-347
11. Bekker, M., Alexeeva, S., Laan, W., Sawers, G., Teixeira de Mattos, J., and Hellingwerf,
K. (2010) The ArcBA two-component system of escherichia coli is regulated by the redox
state of both the ubiquinone and the menaquinone pool J. Bacteriol. 192, 746-754.
12. https://www.genomics.agilent.com/files/manual/200249.pdf
13. Kirubakaran, S. I. Sakthivel, M. (2007) Cloning and overexpression of antifungal barley
chitinase gene in Escherichia coli. Protein Expr. and Purific. 52, 159-166.
20
Appendix 1: Sequences of the primers used during this project
pET28YtvAFW
5’ccccatatggctagttttcaatcatttgggata 3’
NdeI
pET28Sln1pRV
Gcatatcagggaaagaaaaataacaaagctcgagccc
To keep the reading frame for C terminal histag, stop codon
was removed and one g before Ctcgag (reading frame gct gag)
gct gives alanine. The same in all RV primers.
For order rev/comp 5’gggctcgagctttgttatttttctttccctgatatgC 3’
Tcaagtttagtgttgctaaaagcatcgctcgagccc
XhoI
pET28sln1pHKRV
XhoI
Rev/comp
5’ gggctcgagcgatgcttttagcaacactaaacttga 3’
Tm
without
restriction
51
Full 61
Tm
without
restriction
54
Full 62
Tm
without
restriction
55
Full 66
pET28sskn7FW
ccccatatgAGCTTTTCCACCATAAATAGCAACG
NdeI
Tm
without
restriction
57
Full 63
pET28sskn7RV
CTACACTTCAAGAAAACCAGCTATCAgctcgagccc
5’ gggctcgagcTGATAGCTGGTTTTCTTGAAGTGTAG 3’
XhoI
Tm
without
restriction
55
Full 66
JcHpQE30_FW
5’cccgaaaagtgccacctg3’
Bam
HI
JcHpQE30_RV
5’ccgagcgttctgaacaaatcc3’
HindI
II
JBS60/T7_FW
TAATACGACTCACTATAGGG
NdeI
JBS61/T7_RV
GCTAGTTATTGCTCAGCGG
XhoI
pET28ssk1FW
ccccatatgCTCAATTCTGCGTTACTGTGG
NdeI
Tm
without
restriction
58
Tm
without
restriction
64
Tm
without
restriction
56
Tm
without
restriction
58
Tm
without
restriction
56
Full 63
21
pET28ssk1RV
CTCGCCCACTCAAATAGAATTGgctcgagccc
XhoI
Tm
without
restriction
54
Full 67
SalI
Tm
without
restriction
61
5’ gggctcgagcCAATTCTATTTGAGTGGGCGAG 3’
pQE30Sln1pRV
5’Ggg gtcgac tcatttgttatttttctttccctg3’
22
Appendix 2: Protocol PCR with Pwo polymerase
(J.B. van der Steen 18.05.2010)
Pwo polymerase (Roche) is well-suited for accurate amplifications of large fragments.
Reaction mix
Mix the components according to the following table. Reaction volumes of 25-100 μL usually give the
best results.
Component
Stock
For 25 μL For 50 μL For 100 μL
10x buffer + MgSO4 Provided with enzyme
2.5 μL
5.0 μL
10.0 μL
FW/RV primers
10 pmol/μL
1.0 μL
2.0 μL
4.0 μL
10 mM dNTP mix
Fermentas
0.313 μL 0.625 μL
1.25 μL
Water ‡
19.0 μL
38.0 μL
76.0 μL
DNA ‡
1.0 μL
2.0 μL
4.0 μL
Pwo polymerase
Roche
0.25 μL
0.5 μL
1.0 μL
‡ The amount of DNA will vary with the concentration. Adjust the amount of DNA and the amount of
water at will.
Making master mixes without the DNA is usually most convenient. The amounts required for some
regularly used master mixes are shown below (values in μL).
Samples
2 x 25 μL
3 x 25 μL
4 x 25 μL
5 x 25 μL
6 x 25 μL
7 x 25 μL
8 x 25 μL
9 x 25 μL
10 x 25 μL
2 x 50 μL
3 x 50 μL
4 x 50 μL
5 x 50 μL
6 x 50 μL
7 x 50 μL
8 x 50 μL
9 x 50 μL
10 x 50 μL
Buffer
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
Primers
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0
20.0
dNTPs
0.63
0.94
1.25
1.57
1.88
2.19
2.50
2.82
3.13
1.25
1.88
2.50
3.13
3.75
4.38
5.00
5.63
6.25
Water
38.0
57.0
76.0
95.0
114.0
133.0
152.0
171.0
190.0
76.0
114.0
152.0
190.0
228.0
266.0
304.0
342.0
380.0
Pwo
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Cycler program
For products smaller than 1 kb use an extension time of 1:00 min. For larger products, add 1:00 min
per kb.
Cycle 1
Cycle 2
1x
10x
Cycle 3
25x
Cycle 4
Cycle 5
1x
1x
95°C
95°C
Tm-5°C
72°C
95°C
Tm
72°C
72°C
4°C
05:00 min
00:30 min
00:30 min
02:00 min
00:30 min
00:30 min
02:00 min
08:00 min

23
Appendix 3: Protocol preparation of competent cells
(J.B. van der Steen 18.05.2010)
1.
Make an overnight culture in 20 mL LB medium with the appropriate antibiotics.
2.
Add part of the overnight culture to 1 L fresh LB medium to obtain an OD600 of 0.1-0.2.
Note: Usually the entire overnight culture can be used.
3.
When the cells have reached an OD600 of 0.5-1.0, place them on ice for 15-30 minutes.
4.
Centrifuge for 15 minutes at 5000 rpm in a Sorval RC-5 centrifuge with a GS-3 rotor.
5.
Re-suspend the pellets in a total of 1 L cold, sterile NANOpure water and centrifuge again.
6.
Re-suspend the pellets in a total of 0.5 L cold, sterile NANOpure water and centrifuge again.
7.
Re-suspend the pellets in a total of 20 mL cold, sterile 10% glycerol and transfer the cell
suspension to a 50 mL Greiner tube. Now centrifuge for 15 minutes at 4000 rpm (the maximum
speed).
8.
Re-suspend the pellet in cold, sterile 10% glycerol to a final volume of 2-3 mL. Distribute the cells
in 40 μL portions over 1.5 mL eppendorf cups, flash freeze them in liquid nitrogen and store them
at -80°C.
24
Appendix 4: Protocol transformation
(J.B. van der Steen 18.05.2010)
Electrocompetent cells
1.
Thaw the competent cells on ice. Make sure the DNA is dissolved in a low-ionic strength buffer
(preferably water). Add 1-2 μL of your DNA to 40 μL of competent cells, mix by tapping the tube
with a finger and place the suspensions on ice for about 1 minute.
Note: Use 1-2 μL for a QuickChange reaction or a ligation, but do not use more than 0.5-1 ng
of plasmid DNA.
2.
Set the BIO-RAD E. coli Pulser at 2.5 kV.
Note: This should result in a pulse of 4-5 ms.
Note: If a BIO-RAD Gene Pulser is available, set it at 25 μF and 200 Ω.
3.
Transfer the cells to a pre-cooled electroporation cuvette (0.2 cm). Make sure the cell suspension
is at the bottom by tapping the cuvette against the table.
Note: Make sure to dry the cuvette before proceeding to prevent sparking.
4.
Place the cuvette in the Pulser. Pulse once at the described settings. Add 1 mL SOC medium as
soon as possible after the pulse. Re-suspend the cells and transfer them back to the 1.5 mL
eppendorf cup.
Note: The rapid addition of SOC medium is essential for the recovery of transformants.
5.
Incubate the cells at 37°C for 1 hour, shaking.
Note: The easiest way is to tape the tubes to the bottom of a 37°C, 250 rpm incubator.
Alternatively an Eppendorf Thermomixer can be used.
Note: Without shaking the transformation should still succeed, albeit with a much lower
efficiency.
6.
Centrifuge the cells for 10-30 seconds at 8000 rpm in a table-top centrifuge and discard the
supernatant. Re-suspend the cells and plate them on an appropriate plate.
Chemically competent cells
1.
Add an appropriate amount of DNA to 200 μL cells and incubate for 30 min on ice. Include a
negative control without any DNA.
Note: The transformation efficiency should be so high that it is suitable to dilute plasmid
purifications to less than a 100 ng. Alternatively, fewer cells can be used.
2.
Heat-shock the cells for 1 min in a water bath at 42°C. Immediately transfer the cells to ice and
incubate for 1-2 min.
3.
Add 800 μL pre-warmed LB and incubate for 45 min at 37°C, 250 rpm.
Note: Instead of LB, other rich media such as TSB also work.
4.
Plate 25-100 μL on an LB plate with the appropriate marker, gently spin down the rest and plate
this on a different plate. Incubate the plates overnight at 37°C.
25
Appendix 5: Protocol PCR with TAQ polymerase
(J.B. van der Steen 18.05.2010)
TAQ polymerase (Fermentas) is best-suited for control-PCRs. This protocol works well for colonyPCRs on E. coli and for PCRs on 0.5 μL of an overnight culture of B. subtilis.
Reaction mix
Mix the components according to the following table. Reaction volumes of 25 μL usually give the best
results.
Component
Stock
For 10 μL For 25 μL For 50 μL For 100 μL
10x buffer – MgCl2 + KCl Provided with enzyme
1.0 μL
2.5 μL
5.0 μL
10.0 μL
25 mM MgCl2
Provided with enzyme
0.6 μL
1.5 μL
3.0 μL
6.0 μL
FW/RV primers
10 pmol/μL
0.2 μL
0.5 μL
1.0 μL
2.0 μL
10 mM dNTP mix
Fermentas
0.2 μL
0.5 μL
1.0 μL
2.0 μL
Water ‡
7.7 μL 19.25 μL
38.5 μL
77.0 μL
TAQ polymerase
Fermentas
0.1 μL
0.25 μL
0.5 μL
1.0 μL
‡ This protocol does not take the addition of DNA into account. Subtract the amount of DNA added
from the amount of water used. It is fine to add 1 μL DNA without changing the values.
Making master mixes without the DNA is usually most convenient. The amounts required for some
regularly used master mixes are shown below (values in μL). Note that it is possible to lower the
amount of TAQ polymerase per reaction to as little as 0.15 μL in 25 μL.
Samples
2 x 25 μL
3 x 25 μL
4 x 25 μL
5 x 25 μL
6 x 25 μL
7 x 25 μL
8 x 25 μL
9 x 25 μL
10 x 25 μL
20 x 25 μL
30 x 25 μL
Buffer
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
50.0
75.0
MgCl2
3.0
4.5
6.0
7.5
9.0
10.5
12.0
13.5
15.0
30.0
45.0
Primers
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
10.0
15.0
dNTPs
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
10.0
15.0
Water
38.50
57.75
77.00
96.25
115.50
134.75
154.00
173.25
192.50
385.00
577.50
TAQ
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
5.00
7.50
Cycler program
For products smaller than 1 kb use an extension time of 1:00 min. For larger products, add 1:00 min
per kb.
Cycle 1
Cycle 2
1x
10x
Cycle 3
25x
Cycle 4
Cycle 5
1x
1x
95°C
95°C
Tm-5°C
72°C
95°C
Tm
72°C
72°C
4°C
02:00 min
00:30 min
00:30 min
02:00 min
00:30 min
00:30 min
02:00 min
08:00 min

26
Appendix 6: Mobility shift detection of phosphorylated ArcA on Phos-tag
SDS PAGE.
(A. Bury 27.08.2012)
Required solutions:
 5 µM Phos-Tag AAL solution in 3% (v/v) MeOH
Dissolve oily product Phos-tag AAL in 0.1mL MeOH and dilute with 3.2 mL distilled water by pipetting.
If a trace amount of insoluble white powder appears (impurities) it can be separated by centrifuging
(2000xg, 10 min). Store the solution at 4°C, in dark for not more than 6 months.


10 mM MnCl2
o
MnCl2(H2O)4 (FW 197.9 g/mol)
0.1 mg
o
Distilled water
50 mL
MnCl2 solution is stable for at least 6 months.
Lower gel buffer:
1.5 M Tris-HCl pH 8.8, 0.4% w/v SDS, for 100 mL,:

o
Trizma Base
18.171 g
o
SDS
0.4 g
o
Adjust the pH with 6 M HCl.
Upper gel buffer:
0.5 M Tris-HCl pH 6.5, 0.4% w/v SDS, for 100 mL:
o
Trizma Base
6.057 g
o
SDS
0.4 g
o
Adjust the pH with 6 M HCl.

Loading Buffer:
o
o
o
o
o
o

o
o
o
o

o
o
o

bromophenol blue
10 mg
SDS
0.9 g
Glycerol (99.5%)
3
Upper Gel buffer
3.9 mL
β- mercaptoethanol
1.5 mL
Make up the volume to 10 ml in water
10x TGS electrophoresis buffer, for 1 liter:
Trizma Base
30.3 g
Glycine
144 g
SDS
10 g
Results in solution with an approximate pH of 8.3.
10% APS, for 2 mL (FW 228.2 g/mol):
ammonium persulfate
in 2 mL water.
0.2 g
Store the APS in portions of 100 μL at -20°C.
Resolving gel solution:
7 mL; 50 µM Phos-tag, 100 µM MnCl2 10% acrylamide
27
o
o
o
o
o
o
o

30% acrylamide/bis solution 37.5:1 (Biorad) A777
2.33 mL
lower gel buffer (pH 8.8)
1.75 mL
5 mM Phos-tag
70 µL
10 mM MnCl2
70 µL
Distilled water
2.725 mL
TEMED
20 µL
10% APS
35 µL
Stacking gel solution: 2 mL; 4.5% acrylamide
o
o
o
o
o
30% acrylamide/bis solution 37.5:1 (Biorad) A777
0.3 mL
Upper gel buffer (pH 6.8)
0.5 mL
Distilled water
1.145 mL
TEMED
20 µL
10% APS
35 µL
 Staining:
Required solutions


Coomassie Brilliant Blue staining solution, for 500 mL:
o 200 mL methanol (40%).
o 50 mL acetic acid (10%).
o 250 mL water.
o Add 0.125 g Coomassie Brilliant Blue (CBB).
De-staining solution, for 1 liter:
o 400 mL methanol (40%).
o 100 mL acetic acid (10%).
o 500 mL water.
o Note: this is the staining solution without CBB.
28
Appendix 7: Complete nucleotide sequence of C2A aligned with C2A
transformed in E. coli XL1-Blue (using ClustalW2)
Forward primer: JBS60/T7_FW
C2A
Transformed_C2A
-----------------------------------------------------------GGTGGACGGTACATTCCCCTCTAGAATAATTTTGTTTAACTTTAAGAAGGAGATATACCA 60
C2A
Transformed_C2A
-----------------------------------------------------------A 1
TGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATA 120
*
C2A
Transformed_C2A
TGGCTAGTTTTCAATCATTTGGGATACCAGGACAGCTGGAAGTCATCAAAAAAGCACTTG 61
TGGCTAGTTTTCAATCATTTGGGATACCAGGACAGCTGGAAGTCATCAAAAAAGCACTTG 180
************************************************************
C2A
Transformed_C2A
ATCACGTGCGAGTCGGTGTGGTAATTACAGATCCCGCACTTGAAGATAATCCTATTGTCT 121
ATCACGTGCGAGTCGGTGTGGTAATTACAGATCCCGCACTTGAAGATAATCCTATTGTCT 240
************************************************************
C2A
Transformed_C2A
ACGTAAATCAAGGCTTTGTTCAAATGACCGGCTACGAGACCGAGGAAATTTTAGGAAAGA 181
ACGTAAATCAAGGCTTTGTTCAAATGACCGGCTACGAGACCGAGGAAATTTTAGGAAAGA 300
************************************************************
C2A
Transformed_C2A
ACTGTCGCTTCTTACAGGGGAAACACACAGATCCTGCAGAAGTGGACAACATCAGAACCG 241
ACTGTCGCTTCTTACAGGGGAAACACACAGATCCTGCAGAAGTGGACAACATCAGAACCG 360
************************************************************
C2A
Transformed_C2A
CTTTACAAAATAAAGAACCGGTCACCGTTCAGATCCAAAACTACAAAAAAGACGGAACGA 301
CTTTACAAAATAAAGAACCGGTCACCGTTCAGATCCAAAACTACAAAAAAGACGGAACGA 420
************************************************************
C2A
Transformed_C2A
TGTTCTGGAATGAATTAAATATTGATCCAATGGAAATAGAGGATAAAACGTATTTTGTCG 361
TGTTCTGGAATGAATTAAATATTGATCCAATGGAAATAGAGGATAAAACGTATTTTGTCG 480
************************************************************
C2A
Transformed_C2A
GAATTCAGAATGATATCACCAAGCAAAAAGAATATGCTCTTCTAGAAGAAAGAGTTAGGG 421
GAATTCAGAATGATATCACCAAGCAAAAAGAATATGCTCTTCTAGAAGAAAGAGTTAGGG 540
************************************************************
C2A
Transformed_C2A
CGAGGACAAAACAACTCGAAGCTGCCAAGATTGAGGCAGAGGCCGCAAATGAAGCAAAAA 481
CGAGGACAAAACAACTCGAAGCTGCCAAGATTGAGGCAGAGGCCGCAAATGAAGCAAAAA 600
************************************************************
C2A
Transformed_C2A
CCGTCTTTATTGCCAATATTTCGCACGAATTGAGAACGCCTTTAAATGGTATTCTGGGTA 541
CCGTCTTTATTGCCAATATTTCGCACGAATTGAGAACGCCTTTAAATGGTATTCTGGGTA 660
************************************************************
C2A
Transformed_C2A
TGACGGCTATTTCAATGGAAGAAACCGATGTTAACAAAATAAGAAATAGTTTAAAACTCA 601
TGACGGCTATTTCAATGGAAGAAACCGATGTTAACAAAATAAGAAATAGTTTAAAACTCA 720
************************************************************
C2A
Transformed_C2A
TTTTTAGATCAGGTGAGCTTTTGCTTCATATTCTAACGGAATTGTTAACTTTTTCCAAAA 661
TTTTTAGATCAGGTGAGCTTTTGCTTCATATTCTAACGGAATTGTTAACTTTTTCCAAAA 780
************************************************************
C2A
Transformed_C2A
ACGTTCTTCAAAGAACGAAACTGGAGAAAAGAGATTTTTGCATTACCGATGTTGCCTTAC 721
ACGTTCTTCAAAGAACGAAACTGGAGAAAAGAGATTTTTGCATTACCGATGTTGCCTTAC 840
************************************************************
C2A
Transformed_C2A
AAATAAAATCGATATTTGGTAAAGTTGCAAAGGATCAGCGTGTTCGTCTTTCAATATCAT 781
AAATAAAATCGATATTTGGTAAAGTTGCAAAGGATCAGCGTGTTCGTCTTTCAATATCAT 900
************************************************************
C2A
Transformed_C2A
TGTTTCCTAATTTGATAAGGACAATGGTTCTTTGGGGTGATTCCAACAGAATTATTCAAA 841
TGTTTCCTAATTTGATAAGGACAATGGTTCTTTGGGGTGATTCCAACAGAATTATTCAAA 960
************************************************************
C2A
Transformed_C2A
TTGTGATGAATCTAGTGTCCAATGCACTAAAGTTCACCCCTGTAGATGGTACCGTTGATG 901
TTGTGATGAATCTAGTGTCCAATGCACTAAAGTTCACCCCTGTAGATGGTACCGTTGATG 1020
************************************************************
29
C2A
Transformed_C2A
TAAGAATGAAACTGTTGGGTGAATACGACAAAGAATTAAGCGAGAAGAAGCAATACAAAG 961
TAAGAATGAAACTGTTGGGTGAATACGACAAAGAATTAAGCGAGAAGAA-CCATACAAAG 1079
************************************************* * ********
C2A
Transformed_C2A
AAGTGTATATCAAAAAAGGGACAGAAGTAACCGAAAATTTAGAAACTACAGATAAATACG 1021
AAGTG-ATATCAAAAAAGGGACAGAAGTAACCGAAAATTTAGAA-CTACAGATAA-TACG 1136
***** ************************************** ********** ****
Backward primer: JBS61/T7_RV
C2A
Transformed_C2A
CAAATAAAATCGATATTTGGTAAAGTTGCAAAGGATCAGCGTGTTCGTCTTTCAATATCA 780
-AAATAAAATCGATATTTG-TAAAGT-GCAAAGGATCAGCGTGTTCGTCTT-CAATATCA 270
****************** ****** ************************ ********
C2A
Transformed_C2A
TTGTTTCCTAATTTGATAAGGACAATGGTTCTTTGGGG-TGATTCCAACAGAATTATTCA 839
T-GTTTCCTAATTTGATAAGGACAATGGTTCTTTGGGGGTGATTCCAACAGAATTATTCA 329
* ************************************ *********************
C2A
Transformed_C2A
AATTGTGATGAATCTAGTGTCCAATGCACTAAAGTTCACCCCTGTAGATGGTACCGTTGA 899
AATTGTGATGAATCTAGTGTCCAATGCACTAAAGTTCACCCCTGTAGATGGTACCGTTGA 389
************************************************************
C2A
Transformed_C2A
TGTAAGAATGAAACTGTTGGGTGAATACGACAAAGAATTAAGCGAGAAGAAGCAATACAA 959
TGTAAGAATGAAACTGTTGGGTGAATACGACAAAGAATTAAGCGAGAAGAAGCAATACAA 449
************************************************************
C2A
Transformed_C2A
AGAAGTGTATATCAAAAAAGGGACAGAAGTAACCGAAAATTTAGAAACTACAGATAAATA 1019
AGAAGTGTATATCAAAAAAGGGACAGAAGTAACCGAAAATTTAGAAACTACAGATAAATA 509
************************************************************
C2A
Transformed_C2A
CGATCTTCCAACTTTATCGAACCATAGGAAAAGTGTCGATTTAGAATCCAGCGCTACTTC 1079
CGATCTTCCAACTTTATCGAACCATAGGAAAAGTGTCGATTTAGAATCCAGCGCTACTTC 569
************************************************************
C2A
Transformed_C2A
CCTAGGAAGTAATAGAGACACTTCGACAATTCAGGAAGAGATAACAAAAAGAAATACTGT 1139
CCTAGGAAGTAATAGAGACACTTCGACAATTCAGGAAGAGATAACAAAAAGAAATACTGT 629
************************************************************
C2A
Transformed_C2A
TGCGAATGAAAGTATCTATAAGAAAGTGAATGACAGGGAAAAAGCTTCGAATGATGATGT 1199
TGCGAATGAAAGTATCTATAAGAAAGTGAATGACAGGGAAAAAGCTTCGAATGATGATGT 689
************************************************************
C2A
Transformed_C2A
ATCTTCTATAGTATCAACAACTACCAGCTCGTATGATAACGCTATCTTCAATAGTCAGTT 1259
ATCTTCTATAGTATCAACAACTACCAGCTCGTATGATAACGCTATCTTCAATAGTCAGTT 749
************************************************************
C2A
Transformed_C2A
CAATAAAGCACCTGGCTCAGATGATGAAGAAGGTGGTAACCTAGGAAGACCTATCGAAAA 1319
CAATAAAGCACCTGGCTCAGATGATGAAGAAGGTGGTAACCTAGGAAGACCTATCGAAAA 809
************************************************************
C2A
Transformed_C2A
CCCCAAAACGTGGGTTATTTCTATTGAAGTGGAAGACACTGGGCCTGGTATTGACCCTTC 1379
CCCCAAAACGTGGGTTATTTCTATTGAAGTGGAAGACACTGGGCCTGGTATTGACCCTTC 869
************************************************************
C2A
Transformed_C2A
CTTACAAGAATCTGTATTTCATCCATTTGTTCAAGGTGATCAAACATTGTCCAGGCAATA 1439
CTTACAAGAATCTGTATTTCATCCATTTGTTCAAGGTGATCAAACATTGTCCAGGCAATA 929
************************************************************
C2A
Transformed_C2A
TGGTGGTACTGGCTTAGGTCTATCAATCTGTAGACAGTTAGCAAATATGATGCATGGAAC 1499
TGGTGGTACTGGCTTAGGTCTATCAATCTGTAGACAGTTAGCAAATATGATGCATGGAAC 989
************************************************************
C2A
Transformed_C2A
GATGAAATTAGAGTCGAAAGTAGGTGTTGGTAGTAAATTCACTTTTACCTTGCCATTAAA 1559
GATGAAATTAGAGTCGAAAGTAGGTGTTGGTAGTAAATTCACTTTTACCTTGCCATTAAA 1049
************************************************************
C2A
Transformed_C2A
TCAAACTAAAGAGATCAGTTTTGCAGATATGGAGTTTCCTTTTGAGGACGAATTTAATCC 1619
TCAAACTAAAGAGATCAGTTTTGCAGATATGGAGTTTCCTTTTGAGGACGAATTTAATCC 1109
************************************************************
C2A
Transformed_C2A
TGAGAGTAGAAAGAACAGAAGAGTCAAGTTTAGTGTTGCTAAAAGCATC----------- 1668
TGAGAGTAGAAAGAACAGAAGAGTCAAGTTTAGTGTTGCTAAAAGCATCGCTCGAGCACC 1169
*************************************************
C2A
Transformed_C2A
--------------------------------------------------------ACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGAAGCTGATTTCCA 1226
30
Appendix 8: FPLC graphs of the Ni-column purification with imidazole
gradient
Attempt 1:
ArcA
Attempt 2:
Skn7Rec
Attempt 3:
Skn7Rec
Attempt 7:
Skn7Rec
Attempt
11:
Skn7
Attempt
16:
C2A
Table 5: Different conditions for the overproduction of the proteins Skn7Rec, Skn7, C2A and Ssk1Rec
Attempt
Protein
Temperature
ArcA
Skn7Rec
Skn7Rec
Skn7Rec
Skn7Rec
Skn7Rec
Skn7Rec
IPTG
concentration
1 mM
1 mM
1 mM
0.5 mM
1 mM
0.5 mM
1 mM
RT
RT
37˚C
RT
30˚C
30˚C
RT
Time of IPTG
overexpression
o/n
o/n
3h
3h
o/n
o/n
o/n
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Skn7Rec
0.5 mM
RT
o/n
Skn7
Skn7
1 mM
0.5L 1mM
0.5L 20 μM
1 mM
0.5L 1 mM
0.5L 20 μM
1 mM
RT
RT
RT
RT
RT
RT
RT
o/n
o/n
o/n
o/n
o/n
o/n
o/n
C2A
C2A
Ssk1Rec
Lysing method
Sonication
Sonication
French press
French press
Sonication
Sonication
Sonication
French press
Sonication
French press
Sonication
Sonication
Sonication
Sonication
Sonication
Sonication
Sonication
Concentration
(BSA BioRad assay)
25 μM
14 μM
3 μM
5 μM
6 μM
7 μM
-
31
Appendix 9: Complete nucleotide sequence of Ssk1 aligned with Ssk1
transformed in E. coli XL1-Blue (using CluctalW2)
Forward primer: JcHpQE30_FW
Ssk1
Transformed_Ssk1
-----------------------------------------------------------GGGAAATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC 60
Ssk1
Transformed_Ssk1
-----------------------------------------------------------TTCACCTCGAGAAATCATAAAAAATTTATTTGCTTTGTGAGCGGATAACAATTATAATAG 120
Ssk1
Transformed_Ssk1
-----------------------------------------------------------ATTCAATTGTGAGCGGATAACAATTTCACACAGAATTCATTAAAGAGGAGAAATTAACTA 180
Ssk1
Transformed_Ssk1
-------------------------------------ATGCTCAATTCTGCGTTACTGTG 23
TGAGAGGATCGCATCACCATCACCATCACGGATCCGCATGCTCAATTCTGCGTTACTGTG 240
***********************
Ssk1
Transformed_Ssk1
GAAGGTTTGGCTACGAATAGACAACTCCACTGATGAAGTAAACCAACCAATTGCTGTACA 83
GAAGGTTTGGCTACGAATAGACAACTCCACTGATGAAGTAAACCAACCAATTGCTGTACA 300
************************************************************
Ssk1
Transformed_Ssk1
GTTCGATGAAATAGATACTGTTGATGATTTGAAGAGCAGGTTTTTTCAGAAACTGAGTTC 143
GTTCGATGAAATAGATACTGTTGATGATTTGAAGAGCAGGTTTTTTCAGAAACTGAGTTC 360
************************************************************
Ssk1
Transformed_Ssk1
GACTCGATGGCGAGAAATTAACGATAATGCTTCCATTGCAATAGGCCTCTACGCACCTAA 203
GACTCGATGGCGAGAAATTAACGATAATGCTTCCATTGCAATAGGCCTCTACGCACCTAA 420
************************************************************
Ssk1
Transformed_Ssk1
ATTTGACAATCAAGCCGACAATACCAGTAGTAACAACACTAACGATAATAGTTGTCGAAG 263
ATTTGACAATCAAGCCGACAATACCAGTAGTAACAACACTAACGATAATAGTTGTCGAAG 480
************************************************************
Ssk1
Transformed_Ssk1
TAAGAGTAACGGTGCTGGAAGTGGCGCCAACCTTTCCGTTAATAGCAATACCAAGAGTTC 323
TAAGAGTAACGGTGCTGGAAGTGGCGCCAACCTTTCCGTTAATAGCAATACCAAGAGTTC 540
************************************************************
Ssk1
Transformed_Ssk1
AGTGAGCCCCACAGCAGGATCATTTGGTCTTTCAAAAGACCTTGCAAAGGACAGGAATGT 383
AGTGAGCCCCACAGCAGGATCATTTGGTCTTTCAAAAGACCTTGCAAAGGACAGGAATGT 600
************************************************************
Ssk1
Transformed_Ssk1
TCTCCAGCATCCTAAACCTACGCAGAAAAGAGGAGCATTATACGACGCCTTTGCCGCCGT 443
TCTCCAGCATCCTAAACCTACGCAGAAAAGAGGAGCATTATACGACGCCTTTGCCGCCGT 660
************************************************************
Ssk1
Transformed_Ssk1
GCCGACAGTGGCCGCGACTACCAATGTGGATTTTCCTCCCAACGAGGCGCCAATGCTAAG 503
GCCGACAGTGGCCGCGACTACCAATGTGGATTTTCCTCCCAACGAGGCGCCAATGCTAAG 720
************************************************************
Ssk1
Transformed_Ssk1
CCCGCAAAGACCATACTCTACTAGTCCTAAACAGTTTCCAGCAACAACTAAAAGTCCGTT 563
CCCGCAAAGACCATACTCTACTAGTCCTAAACAGTTTCCAGCAACAACTAAAAGTCCGTT 780
************************************************************
Ssk1
Transformed_Ssk1
ACTGCGATTTGCCTCAGTCTCACCCTACCCTAAATTTCATTCTGATAATCAAATTATGGC 623
ACTGCGATTTGCCTCAGTCTCACCCTACCCTAAATTTCATTCTGATAATCAAATTATGGC 840
************************************************************
Ssk1
Transformed_Ssk1
ATCAGCTGGTCTTACATACGTCTCACCGCATAATAAAAATAAATACACAAGGCCGTTGAT 683
ATCAGCTGGTCTTACATACGTCTCACCGCATAATAAAAATAAATACACAAGGCCGTTGAT 900
************************************************************
Ssk1
Transformed_Ssk1
TAGAAAAGGTTTAAATTTTACCACAGAATCAGTTAATGATTGCACTTATAAAATCATCTT 743
TAGAAAAGGTTTAAATTTTACCACAGAATCAGTTAATGATTGCACTTATAAAATCATCTT 960
************************************************************
Ssk1
Transformed_Ssk1
TGAACCGGATGAATTGGCTATTAACATATATAAGGAACTATTCGGAACCATGGGTTCCCA 803
TGAACCGGATGAATTGGCTATTAACATATATAAGGAACTATTCGGAACCATGGGTTCCCA 1020
************************************************************
32
Ssk1
Transformed_Ssk1
ACCTGCATCGCAGCCTTTGCTGATATTTTCGAATGTTAATTTACGCCAGGATGTACCGCC 863
ACCTGCATCGCAGCCTTTGCTGATATTTTCGAATGTTAATTTACGCCAGGATGTACCGCC 1080
************************************************************
Ssk1
Transformed_Ssk1
TTTAGATATCTTAAATGTTGTAGACTATGTTCCTACGAATGAAGAAATTTCGCAGCAGAA 923
TTTAGATATCTTAAATGTTGTAGACTATGTTCCTACGAATGAAGAAATTTCGCAGCAGAA 1140
************************************************************
Ssk1
Transformed_Ssk1
AACTCAACCAACAGACCATGGGG-CCGTTGGTGTTTTTCATCTAGACGACCATATTTCTC 982
AACTCAACCAACAGACCATGGGGGCCGTTGGTGGTTTTCATCTAGACGACCATATTTCTC 1200
*********************** ********* **************************
Ssk1
Transformed_Ssk1
CGGGCGAACAAGGTCTTAAGCAAACAATTGGTGATAAAGCAGATCTTAAAGGTAAAGATG 1042
CGGGCGAACAAGGGCTTAAGCAAACAATTGGTGATAAACCAGATCCTAAAGGGAAAGATG 1260
************* ************************ ****** ****** *******
Backward primer: JcHpQE30_RV
Ssk1
Transformed_Ssk1
AATGAAGAAATTTCGCAGCAGAAAACTCAACCAACAGACCATGGGGCCGTTGGTGTTTTT 960
AAAGAAG-AATTTCGGAGCAG-AAACTCAACCAACAGCCCA-GGGGCCGTTGGGG-TTTT 233
**:**** ******* ***** ***************.*** *********** * ****
Ssk1
Transformed_Ssk1
CATCTAGACGACCATATTTCTCCGGGCGAACAAGGTCTTAAGCAAACAATTGGTGATAAA 1020
CATCTAGAGGACCATATTT-TCCGGGCGAACAAGGTCTTAAGCAAACAATTGGTGATAAA 292
******** ********** ****************************************
Ssk1
Transformed_Ssk1
GCAGATCTTAAAGGTAAAGATGGCAATAGCAGCCCTCAGGAATTTAAATTAATAACTGAT 1080
GCAGATCTTAAAGGTAAAGATGGCAATAGCAGCCCTCAGGAATTTAAATTAATAACTGAT 352
************************************************************
Ssk1
Transformed_Ssk1
GAAGAGCAATTGAGAAGAGCGTCACAAGAACTGAAGGATGAGGAAAAGGATGCCGAGTCT 1140
GAAGAGCAATTGAGAAGAGCGTCACAAGAACTGAAGGATGAGGAAAAGGATGCCGAGTCT 412
************************************************************
Ssk1
Transformed_Ssk1
CCTTGGCAAGCAATCTTGCTGTTACCAAAAGGTTATAAAGGAGGGGTAGATTTTCGAAAT 1200
CCTTGGCAAGCAATCTTGCTGTTACCAAAAGGTTATAAAGGAGGGGTAGATTTTCGAAAT 472
************************************************************
Ssk1
Transformed_Ssk1
AAACCAGTGGCCCACACGGATTCATCTTTCAATAATGAAGACACAATTACTCATTCAGAG 1260
AAACCAGTGGCCCACACGGATTCATCTTTCAATAATGAAGACACAATTACTCATTCAGAG 532
************************************************************
Ssk1
Transformed_Ssk1
TTAGAAGTGAACACCGGATCCCCTTCGCAAGAAAGCGGATCACTTAATGAAGCTGGTATA 1320
TTAGAAGTGAACACCGGATCCCCTTCGCAAGAAAGCGGATCACTTAATGAAGCTGGTATA 592
************************************************************
Ssk1
Transformed_Ssk1
GGCATAACGCAACCCATGTCGGAAGTACAAAGAAGAAAAGAAGACGTTACGCCCGCATCA 1380
GGCATAACGCAACCCATGTCGGAAGTACAAAGAAGAAAAGAAGACGTTACGCCCGCATCA 652
************************************************************
Ssk1
Transformed_Ssk1
CCAATATTAACAAGTAGTCAAACGCCGCATTACTCAAACTCGCTTTATAACGCACCTTTT 1440
CCAATATTAACAAGTAGTCAAACGCCGCATTACTCAAACTCGCTTTATAACGCACCTTTT 712
************************************************************
Ssk1
Transformed_Ssk1
GCTGTTTCCTCTCCACCAGATCCTTTACCAAACCTTTTTACCACCACAAGTGAAAAAGTT 1500
GCTGTTTCCTCTCCACCAGATCCTTTACCAAACCTTTTTACCACCACAAGTGAAAAAGTT 772
************************************************************
Ssk1
Transformed_Ssk1
TTCCCCAAAATTAATGTTTTAATAGTTGAAGACAACGTCATCAACCAAGCTATCTTAGGT 1560
TTCCCCAAAATTAATGTTTTAATAGTTGAAGACAACGTCATCAACCAAGCTATCTTAGGT 832
************************************************************
Ssk1
Transformed_Ssk1
TCCTTTCTGAGGAAACACAAAATCTCATATAAACTGGCTAAAAATGGTCAAGAAGCTGTT 1620
TCCTTTCTGAGGAAACACAAAATCTCATATAAACTGGCTAAAAATGGTCAAGAAGCTGTT 892
************************************************************
Ssk1
Transformed_Ssk1
AATATTTGGAAGGAAGGCGGTCTTCATTTAATATTTATGGATTTACAGCTGCCTGTCTTG 1680
AATATTTGGAAGGAAGGCGGTCTTCATTTAATATTTATGGATTTACAGCTGCCTGTCTTG 952
************************************************************
Ssk1
Transformed_Ssk1
TCTGGTATAGAAGCTGCCAAGCAGATTAGGGACTTCGAAAAACAAAATGGCATTGGCATT 1740
TCTGGTATAGAAGCTGCCAAGCAGATTAGGGACTTCGAAAAACAAAATGGCATTGGCATT 1012
************************************************************
33
Ssk1
Transformed_Ssk1
CAAAAAAGTCTCAATAACTCACACTCCAATCTTGAAAAAGGTACTTCAAAGAGATTCTCT 1800
CAAAAAAGTCTCAATAACTCACACTCCAATCTTGAAAAAGGTACTTCAAAGAGATTCTCT 1072
************************************************************
Ssk1
Transformed_Ssk1
CAGGCGCCCGTGATTATTGTAGCATTGACCGCATCTAACTCTCAGATGGATAAAAGAAAA 1860
CAGGCGCCCGTGATTATTGTAGCATTGACCGCATCTAACTCTCAGATGGATAAAAGAAAA 1132
************************************************************
Ssk1
Transformed_Ssk1
GCACTTCTTTCTGGTTGTAACGACTACCTGACTAAACCAGTGAATTTACACTGGCTTAGT 1920
GCACTTCTTTCTGGTTGTAACGACTACCTGACTAAACCAGTGAATTTACACTGGCTTAGT 1192
************************************************************
Ssk1
Transformed_Ssk1
AAGAAAATTACAGAGTGGGGATGTATGCAAGCCTTGATTGATTTTGACAGCTGGAAGCAG 1980
AAGAAAATTACAGAGTGGGGATGTATGCAAGCCTTGATTGATTTTGACAGCTGGAAGCAG 1252
************************************************************
Ssk1
Transformed_Ssk1
GGAGAAAGCCGGATGACCGACAGTGTTTTGGTTAAATCTCCACAGAAACCTATTGCACCT 2040
GGAGAAAGCCGGATGACCGACAGTGTTTTGGTTAAATCTCCACAGAAACCTATTGCACCT 1312
************************************************************
Ssk1
Transformed_Ssk1
TCCAACCCTCACTCATTCAAACAAGCGACATCTATGACCCCTACACACAGCCCAGTAAGA 2100
TCCAACCCTCACTCATTCAAACAAGCGACATCTATGACCCCTACACACAGCCCAGTAAGA 1372
************************************************************
Ssk1
Transformed_Ssk1
AAAAATTCAAACCTCTCGCCCACTCAAATAGAATTGTGA--------------------- 2139
AAAAATTCAAACCTCTCGCCCACTCAAATAGAATTGTAAAAGCTTAATTAGCTGAGCTTG 1432
*************************************.*
Ssk1
Transformed_Ssk1
---------------------------------------GACTCCTGTGATAGATCCCAGTAAGACCTCAAATCCTCCC 1472
34