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pGLO Lab Report

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Mutagenesis in pGLO Bacteria
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Mutagenesis in pGLO Bacteria
Alissa Eckas and Elizabeth Jorgensen
University of Northern Colorado
Mutagenesis in pGLO Bacteria
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Abstract
In a society where antibiotic resistant super bugs are becoming more and more prevelant
it is important to determine where in the bacterial genome the bacteria is susceptible to
antibiotics and in what circumstances the bacteria will fail to thrive. By using E. coli bacteria
and a Tn5 transposon we were able to test where in the three genetic coding regions the
transposon inserted and mutated. A green colony indicated that the araC gene bound to the GFP
promoter fed by the arabinose sugar present. As a control a plate utilizing only LB broth and no
DNA was plated, in the absence of sugar and E. coli DNA only white colonies were possible as
arabinose is required for the green florescent protein to activate. Further, as it is mutagenized
bacteria that is able to grow in the presence of ampicillin or kanamycin we needed a control to
show that bacteria was in fact present in the samples and capable of producing colonies.
Introduction
Bacterial plasmids have been utilized in genetics research for a broad range of
applications including: functional DNA analysis, gene targeted therapies for applications such as
vaccines and new antibiotics, DNA sequencing and identification of proteins (Choi & Kim,
2009). Plasmids serve many purposes within the bacterial genome, including extending
antibiotic resistance to new cells and promotion of bacterial mating thus creating exponentally
more copies of the bacteria being studied (Pierce, 2014). The pGLO plasmid being utilized for
this experiment contains three distinct genes that affect the growth of the E. coli bacteria
depending on the insertion site of the transposon. A green florescent protein (GFP) has been
inserted into the pGLO plasmid, in the presence of this protein bacterial colonies will glow green
under UV lights (Leatherman, 2018). Also present is a gene that encodes for the AmpR enzyme.
We are able to test for the presence of this enzyme by plating samples on ampicillin containing
Mutagenesis in pGLO Bacteria
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plates and observing growth or lack thereof (Leatherman, 2018). Further for the E. coli bacteria
to grow in the presence of ampicillin the protein beta-lactamase is needed as it acts to inactivate
ampicillin (Leatherman, 2018). The araC gene acts as a regulator of the GFP gene, araC binds to
the promoter in GFP transcription, when arabinose is present araC binds to the promoter leading
to glowing green colonies (Leatherman, 2018).
The Tn5 transposon was used to mutagenize the pGLO plasmid in our study. A
transposon is a short piece of DNA that randomly inserts into the plasmid and can render the
gene non-functional. Transposons are also referred to as “jumping genes” because of their
ability to shift from position to position in the genome causing chromosomal mutations along the
way (Choi & Kim, 2009). The insertion point of the Tn5 transposon in our pGLO plasmids will
be determined by use of electrophoresis testing.
Digest enzymes are used to “cut” the DNA and predict where the transposon inserted into
the genome. By using different restriction enzymes we are able to determine if the transposon
inserted in a region expected based on the plating results. We expect the transposon regions to
appear longer and darker in the mutagenized bacteria.
Methods
Mutagenesis
To facilitate insertion of the Tn5 transposon into the pGLO plasmid, pGLO plasmids
were combined with deionized water, EZ Tn5 10X reaction buffer, 0.1 p mol/microliter Tn5
transposon and EZ-Tn5 transposase. The solution was centrifuged to incorporate all
components. The solution then underwent a series of incubations at 37oC, was recentrifuged and
a stop solution was added. Finally, the solution was heat shocked at 70oC and then stored in a
freezer.
Mutagenesis in pGLO Bacteria
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Transformation
Chilled calcium chloride solution was added to three tubes of chilled and centrifuged E.
Coli cultures. A series of 4 cooling and centrifugation cycles were completed prior to the
addition of DNA. Tube one was the control tube with no DNA added. Wild type pGLO DNA
solution was added to tube two. Tn5 mutagenized pGLO DNA was added to tube 3. The tubes
were then incubated on ice prior to being cooled, heat shocked, cooled again and then incubated
at 37oC for 30 minutes while being shook. Six plates were prepared: lysogeny broth (LB)
without DNA, no DNA and ampicillin, wild type pGLO DNA and ampicillin, wild type pGLO
DNA with ampicillin and arabinose, mutant pGLO DNA with kanamycin and arabinose and
finally wild type pGLO DNA with kanamycin and arabinose. The plates were incubated
overnight at 37oC, after incubation the plates were stored in a refrigerator.
Purification
Two sterile culture tubes were prepared for this step, one with LB and ampicillin, the
second with LB and kanamycin. A wild-type pGLO colony was taken from the arabinose plate
and pipetted into the LB and ampicillin solution. A mutant pGLO colony was taken from the
kanamycin and ampicillin plate and pipetted into the LB and kanamycin solution. The cultures
were incubated in a shaker incubator overnight. A sterile loop was used to obtain a sample from
the kanamycin resistant solution, the loop was then streaked over an ampicillin and arabinose
plate and incubated overnight.
The bacterial cultures were centrifuged to separate the bacterial pellet from the
supernatant, the supernatant was discarded. Buffer solutions P1, P2 and N3 were added to the
pellet in order and pipetted up and down to mix. The solutions were then centrifuged twice
more, the second time in a spin column to allow for the collection of DNA supernatant. Buffer
Mutagenesis in pGLO Bacteria
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PE was then added to the DNA supernatant. The DNA was then centrifuged twice more to
remove any wash buffer. Fifty microliters of deionized water was then added to the DNA
supernatant before centrifuging. The DNA is now located in the spin through portion of the tube.
DNA concentration readings were taken using a Nanodrop. The wild type concentration was
111.2 ng/ul, the mutagenized concentration was 176.5 ng/ul.
DNA Restriction Digests and Gel Electrophoresis
Restriction enzymes Nde I, Nae I, Xmn I, Xae I and Xmn I were used to determine where
the transposon inserted into the bacterial plasmid. Three digest tests were set up and applied to
both the wild type and mutant plasmids. Digest 1 consisted of Nde I and Nae I, digest 2
consisted of Xmn I and Xae I, finally the third digest consisted of Nae I and Xmn I. The
restriction digests were composed of DNA, 10X buffer, the restriction enzyme and deionized
water. The amount of water, enzymes, and 10X buffer were calculated based on the DNA
concentration of the wild type and mutant pGLO. The restriction digests were incubated at 37oC
for 1.5 hours, then the tubes were removed and stored in the freezer.
Agarose gel was prepared using 1X Tae buffer with agarose powder. The agarose
solution was heated to dissolve the gel granules in the buffer and then allowed to cool before
pouring the mixture into a prepared agarose horizontal gel apparatus. The gel was then covered
with 1X Tae buffer. The restriction enzymes were prepared by adding 10X loading dye to each
tube. The restriction enzymes were pipetted into the slots left from removing the comb. A 10uL
1kb ladder molecular weight marker was used as a control. The gel electrophoresis ran until the
dye could be seen halfway down the gel. The gel was then removed, placed in a staining box
and just covered with SYBR green staining mix. The staining box was covered in aluminum foil
to prevent light from affecting the dye reaction.
Mutagenesis in pGLO Bacteria
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Results
To test where the Tn5 transposon inserted into the E. coli bacteria DNA six experimental
plates were set-up as shown in Table 1. The use of plates containing ampicillin without
arabinose sugar, plates with both arabinose and arabinose and finally kanamycin and arabinose
allowed us to predict colonial growth. The results of the plates were not all consistent with what
was expected. Table 2 shows the outcome of our experiment. In the case of the wild type pGLO
with ampicillin we expected to see white colonies but no colonies were present. This may have
been the result of contamination of the plate when preparing the plate or failing to store the
plates upside down during the initial growth period. Additionally, in the wild type pGLO with
ampicillin and arabinose plate we expected to see a green colony because the plasmid inserted
was expected to be ampicillin resistant and the GF protein flourishes in the presence of arabinose
causing the typical green glow. Again the most likely cause of this outcome was contamination
during the initial plating and possibly failing to store the plates upside down. The remaining four
plates had outcomes as expected. The mutant pGLO with kanamycin and arabinose plate
produced few green colonies but colonies were seen. The LB and no DNA and no antibiotics
plate performed the best with a significant coating of white colonies observed. We chose to take
colonies from the mutant pGLO with kanamycin and arabinose plate for the later stages of our
experiment because this plate both contained DNA and showed the expected results.
In preperation for electrophoresis gel testing the DNA samples had to be combined with
deionized water, 10X buffer and the restriction enzymes being used. Tables 3 and 4 show the
specific amounts of each element used to form the eight test samples. The level of 10X buffer
and enzymes were given. The amount of DNA needed was determined by the formula
C1V1=C2V2 where we have a known final concentration 0.5ug, and a known intitial
Mutagenesis in pGLO Bacteria
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concentration per volume ug/uL. The amount of water needed was determined by subtracting
the known amounts from 20uL to react a final volume of 20 uL.
Table 5 shows where we expect the digest restriction enzymes to cut the DNA. Digest
one contained Nde I and Nae I and was expected to cut the DNA at position 1612 for AmpR, and
position 3312 for the non-coding regions between GFP and AmpR and ORI and araC
respectively. Digest two contained Xmn I and Xae I and is expected to cut AmpR at position
5851, cut the non-coding region between GFP and AmpR at position 1286 and finally to cut the
non-coding region between ORI and araC at position 5851. Digest three contained Nae I and
Xmn I, this digest is expected to cut AmpR at position 2874, the non-coding region between GFP
and AmpR and the non-coding region between ORI and araC at position 2807.
Figure one shows the results of our gel electrophoresis tests. The results seem to indicate
that in our first mutant test that the transposon inserted at around position 3,000 which indicates
that the transposon was inserted in the non-coding region between ORI and araC. Our second
mutant digest seems to have inserted around position 6,000 meaning that we expect that this
transposon inserted in the AmpR coding region. Finally our third digest has inconclussive
results, the extended streak of white on the gel seems to suggest that the transposon inserted
between position 5,000 and position 2,000 as we expected this digest to show a change around
position 2,800 it is my conclusion that this digest does not show a valid result but rather may
have been contaminated or the concentration calculations weren’t correct.
Figure two is the genomic mapping of the pGLO plasmid. The blue lines indicate the the
cut sites of the Nde I restriction enzyme at positions 1340, 1574 and 4927. The purple lines
show where the Nae I restriction enzymes cut at positions 3365 and 5318. The green line shows
where we expect the Xma I resrict enzyme to cut at position 2079. Finally the orange lines show
Mutagenesis in pGLO Bacteria
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where we expect the Xmn I enzyme to cut at positions 2144 and 2823.
DNA Added (no DNA, wt,
mutant)
LB and no DNA
No DNA
Wt pGLO
Wt pGLO
Wt pGLO
Mutant pGLO
Type of Plate
No antibiotics
Ampicillin
Ampicillin
Ampicillin and arabinose
Kanamycin and arabinose
Kanamycin and arabinose
Prediction
White colony
No colony
White colony
Green colony
No colony
White or green colony
Table 1: Experimental Plate set-up
DNA Added (no DNA, wt, mutant)
LB and no DNA
No DNA
Wt pGLO
Wt pGLO
Wt pGLO
Mutant pGLO
Type of Plate
No antibiotics
Ampicillin
Ampicillin
Ampicillin and arabinose
Kanamycin and arabinose
Kanamycin and arabinose
Outcome
White colony
No colony
No colony
No colony
No colony
Green colony
Table 2: Experimental Plate Results
Wild-Type pGLO
Water
10X Buffer
DNA: 0.5 ug (ug/uL of stock 111.2)
Enzyme 1 (20 units/ uL)
Enzyme 2 (20 units/uL)
Uncut
13.51 uL
2 uL
4.49 uL
None
None
Digest 1
11.51 uL
2 uL
4.49 uL
1 uL
1 uL
Digest 2
11.51 uL
2 uL
4.49 uL
1 uL
1 uL
Digest 3
11.51 uL
2 uL
4.49 uL
1 uL
1 uL
Digest 2
13.17 uL
2 uL
2.83 uL
1 uL
1 uL
Digest 3
13.17 uL
2 uL
2.83 uL
1 uL
1 uL
Table 3: Wild-Type pGLO Digest Rescription Enzyme Solution Preperation
Mutant pGLO
Water
10X Buffer
DNA: 0.5 ug (ug/uL of stock 176.5)
Enzyme 1 (20 units/ uL)
Enzyme 2 (20 units/uL)
Uncut
15.17 uL
2 uL
2.83 uL
None
None
Digest 1
13.17 uL
2 uL
2.83 uL
1 uL
1 uL
Table 4: Mutant pGLO Digest Rescription Enzyme Solution Preperation
Wild Type pGLO
AmpR
Non-coding region between GFP and AmpR
Non-coding region between ORI and araC
Uncut
5371
5371 & 1221
5372 & 1221
5373 & 1221
As
Expected
Yes
Yes
No
No
Yes
Yes
Mutagenesis in pGLO Bacteria
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Wild Type pGLO
AmpR
Non-coding region between GFP and AmpR
Non-coding region between ORI and araC
Digest #1
1393, 234, 2091, 1262, 391
1393, 234, 2091, 1262, 1612
282, 234, 3312,1262,391
1393, 234, 3312, 1262, 391
Wild Type pGLO
AmpR
Non-coding region between GFP and AmpR
Non-coding region between ORI and araC
Digest #2
65, 679, 4630
65, 79, 5851
1286, 679, 4630
65, 479, 5851
Wild Type pGLO
AmpR
Non-coding region between GFP and AmpR
Non-coding region between ORI and araC
Digest #3
2132, 1586, 1653
2132, 1586, 2874
2132, 2807, 1653
2132, 2807, 2874
Table 5: Expected Transposon Insertion Sites of Digests
Uncut Mutant
Uncut Wild Type
Digest 1 Mutant
Digest 1 Wild Type
Digest 2 Mutant
Digest 2 Wild Type
Digest 3 Mutant
Digest 3 Wild Type
Observed Bands
None
None
3000-3300 and 16001500
None
6000-5500
4900
6000-2000
6000-2000
Table 6: Gel Electrophosesis Results
Expected Bands
None
None
1612 and 3312
None
5851 and 1286
None
2874 and 2807
None
Digest #1 Band
Change
No change
1612
3312
3312
Digest #2 Band
Change
No change
5851
1286
5851
Digest #3 Band
Change
No change
2874
2807
2807
Mutagenesis in pGLO Bacteria
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Figure 1: Electrophoresis Gel Results
Mutagenesis in pGLO Bacteria
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Figure 2: pGLO plasmid Map
Discussion
This experiment yielded mixed results. Two of the experimental plates did not produce
the expected results. We expected to see a white colony form on the plate containing wild type
pGLO and ampicillin. No colonies formed on this plate, the most logical explination for this was
that there was contamination when the condensation was wiped off of the plate lids prior to
Mutagenesis in pGLO Bacteria
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plating. This contamination could have introduced kanamycin to this plate and the wild type
pGLO cannot grow colonies in the presence of kanamycin. Additionally, the plate containing
wild type pGLO with ampicillin and arabinose did not produce colonies where we expected
green colonies to form. Again, the most likely explination is cross contamination from the
kanamycin containing plates when preparing the plates. This was definitely a case of human
error as we didn’t think to use new wipes for each lid and had prepped our plates prior to the
announcement that we should use separate wipes. In retrospect we should have started over with
this step of the experiment as this oversight very likely skewed our results.
The gel electrophoresis results were not the clearist. The first restriction enzyme appears
to show two possible insertion sites. The brightest spot shows around position 3000 and a fainter
spot around position 1500. This is in line with our expected insertion sites of 1612 and 3312.
Because the spots are not as thick and as bright as restrict two it is less likely that the transposons
inserted in these sites or that there was not sufficient DNA to produce a stronger result.
The second restriction digest enzyme produced the clearist result with a very bright spot
occuring around position 6000 and a faint spot occuring around position 2000. These results are
in line with the expected transposon insertion points of 5851 and 1286. This would indicate that
transposon inserted somewhere between the non-coding regions between AmpR and GFP and
AmpR. The very faint bar around position 2000 is what we would expect for a transposon
inserting in the GFP region, as our plate did not produce very many colonies the fainter lines is
as expected because there was less DNA.
The third restriction enzyme was not conclusive. It is difficult to determine if that
transposon spanned a large area or if insufficient stain was used to yield the true results. It is
Mutagenesis in pGLO Bacteria
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possible that the transposon inserted around position 2800 but as there are no truly bright spots it
is doubtful. As this is the non-coding region between AmpR and GFP we would not expect to
see a strong reaction.
It is difficult to say to what degree the results of this experiment were skewed by cross
contamination in the initial set up of this experiment. It is also possible that the needed volumes
of the components that went into the eight digest tubes was not calculated accurately leading to
insufficient DNA used and an excess of water. Further we may not have used enough staining
dye or allowed the reaction to procedure for long enough. When the gel was taken out of the
reaction box it appeared as if the ladder was half way down the gel but it was a best guestimate.
Mutagenesis in pGLO Bacteria
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References
Choi, K. H., & Kim, K. J. (2009). Applications of transposon-based gene delivery system in
bacteria. Journal of microbiology and biotechnology, 19(3), 217-228.
Leatherman, J. 2018. Genetics laboratory manual. Ann Arbor: XanEdu
Pierce, B.A. 2014.Genetics a conceptual approach. New York: W.H. Freeman and Company
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