OPEN BOOK
BIO 510 Mid-year Exam 2011 NAME (Please Print)___________________________
1( 20 pts) NOTE: This question is related to the basic cloning question from last
year and draws directly from our lab experience in cloning, PCR and protein
expression as well as from our class reading.
The sequence of the BFT1 gene is presented below. The bold and shaded region is the
protein coding sequence. Show all steps needed to create a protein-fusion construct
between the coding sequence of BFT1 and the maltose binding protein encoded by
pMAL-p5X (page 345 in the NEB catalog; note that the sequences presented in the
polylinker are already arranged in the correct reading frame. For instance, under the
XmnI site, the GGA, AGG, ATT and TCA sequences represent codons that are in the
same reading frame with the maltose binding protein). Assume that you start with one
microgram of pMAL-p5X and an unlimited supply of the DNA presented below and any
reagents or materials (e.g., enzymes, oligonucleotides) and machinery (e.g., PCR
machine, incubators, etc) that you need. The only exception is that you cannot propose to
chemically (that is, non-enzymatically) synthesize the entire gene. On the sequence
below, the protein-coding region is shaded and the translational termination codon is
underlined (a restriction map is provided below). The perfect clone (i.e., full-credit) is an
in-frame fusion that includes all the BFT1 coding sequence and little else beyond the
minimum sequence needed to fuse it to the maltose binding peptide.
Discuss every enzymatic step you need to perform in detail and state which specific
enzyme you would use, what buffers, length of incubation, etc. Describe any cleanup
steps needed after one step is completed before the next step is initiated. You need not
discuss E. coli competent cell preparation or the transformation itself, simply every step
needed to get to the point where you have DNA to be transformed into E. coli.
BFT1
1
51
101
151
201
251
ATTACGTCGC
AGACTTGGCA
ACCAGAAAAA
ATCTTGAGAC
GCTTGAGAAG
ACCTGTCCTT
AAGACGATAC
AGACAAAAAA
GAGTGGTGAT
AAAAGCAGGC
TTAAAAGCCG
TTATTGG
ATCTTTAACA
ACTTGAAAAA
CCTAAGAAAA
CGCCGCAGAT
AAAAGACGAG
ATGGCTAGAG
ACAAAAGGAC
GAATGGAGTC
GCTAGAAGAG
AAGATAAGCA
GTAATCAAAG
ATGGCTAAGA
AGACGCCGAA
AGGCTGAAAA
GACACTTTAT
Briefly, the easiest way to do this is simply to PCR amplify the BFT1 coding sequence
and drop it into the pMAL-p5X polylinker. This could be done as a blunt-ended PCR
fragment inserted at the XmnI site after trimming the non-encoded 3’ A extension from
the PCR fragment and phosphorylating its (i.e., the PCR fragment’s) 5’ ends. If you use
this blunt-ended strategy, you should dephosphorylate the vector to prevent self-ligation
and you must determine the orientation of fragment insertion after cloning (since only
one direction gives you the appropriate protein fusion product). Alternatively, you can
include restriction sites compatible with the pMAL-p5X polylinker (e.g., XmnI and NotI)
in the BFT1 amplification by adding these to the 5’ ends of the PCR primers. With
either cloning strategy, you have to adjust the PCR primer positions to retain the
translational reading frame from the maltose binding protein through the BFT1 coding
sequence. Alternative (non-PCR) strategies that use existing restriction sites within the
BFT1 for subcloning were acceptable but full credit requires that the full protein coding
sequence be transferred.
2. (4 pts) NOTE: This question is related our lab experience in protein expression as
well as from our class reading and lecture presentations.
Your main goal is to purify large amounts of recombinant protein. Which of the two
strains presented below would you choose for this experiment and why (marker
descriptions on NEB 320-321)?
c. ER3020 McrA+, McrBC-, EcoKR-, EcoKM- , lon-, ompT, sulA-, endA
Only this strain has the mutations needed to stabilize foreign proteins in E. coli (lon,
ompT, sulA).
3. (5 pts) This question is related our lab experience in cloning as well as from our
class reading and lecture presentations. Which enzyme pairs produce DNA with ends
that are always compatible (i.e., ends that can be ligated together)?
A) SspI,
AAT/ATT
D) CviKI-1, RG/CY
C) SalI,
F) XhoI,
G/TCGAC
C/TCGAG
G) BamHI
j) Sau3AI
G/GATCC
/GATC
CLOSED BOOK
1) (3 pts) Note: From radiation lecture and lab activities. The radioisotope we
used to prepare our plus and minus strand RNA hybridization probes was 32P
(note, provide both the element and the atomic number of the isotope). This
isotope is a beta particle emitter and that compared to tritium (3H) is a (high)
energy emission.
2) (4 pts) Three steps relevant to laboratory radioisotope use were suggested in our
radiation training to limit radiation exposure. These were to:
a. Reduce the time of exposure
b. Increase the distance between the worker and the isotope
c. Provide shielding between the worker and the isotope
d. Following steps a through c above results in a use practice abbreviated
ALARA. The letters in ALARA stand for: As Low as Reasonably
Achievable
3) (10 pts) From lectures, lab activities and reading assignments. Which of the
following would be useful for (put letter alongside protein or process): 1)
M13KO7 DNA polymerase D, 2) solubilizing E. coli, 3 A) lacZ gene expression
E, 3) imaging DNA by florescence B, 4) selection of a chitin-binding protein C
A)
D)
B)
C
E)
4) (4 pts) We did an in-class comparison between EtBr and SYBR safe for the
detection of dsDNA. Add one or the other product name for each of the following
blanks. We found that EtBr was more sensitive for detecting low levels of DNA
under the conditions of use. EtBr was much less expensive to use when only the
purchase price of the raw reagent was considered. EtBr has reported as a mutagen.
EtBr must be disposed of as a hazardous material.
5) (10 pts) From lectures, lab activities and reading assignments. Describe in
detail the substrate and describe products of:
a. T4 polynucleotide kinase Substrate: 5’ hydroxyl on RNA or DNA (SS or DS
although 3’ recessed ends are modified slowly) plus ATP; product: 5’
phosphate group
b. Exonuclease III – Substrate: dsDNA with blunt or 5’ extended end; Product:
removes nucleotides in a 3’-5’ manner leaving a partially ss substrate and
released nucleotides
c. RNase H: Substrate: RNA in an RNA/DNA hybrid; Products, ssDNA and
released RNA nucleotides monophosphates and small RNA fragments
d. Shrimp alkaline phosphatase: Substrate: 5’ phosphate on ss or dsDNA or
RNA; Product: released inorganic phosphate & nucleic acid with 5’ hydroxyl
e. M13 K07 DNA polymerase Substrate: dsDNA with an appropriate viral
replication origin plus dNTPs; Product: released ssDNA
f. β-galactosidase; Substrate: lactose (or X-gal); Product: glucose+ galactose (or
chromogenic X-gal metabolite)
g. Reverse transcriptase: Substrate: primed ssRNA plus dNTPs; Product:
Initially, a RNA/DNA hybrid
h. RISC complex: Substrate: single stranded mRNA (or other RNA) target;
Product: cleaved DNA at the site of RISC binding leading to full mRNA
degradation
i. RNase A: Substrate: Single stranded RNA; Products: cleaves adjacent to
pyrimidines, released nucleotides monophosphates and RNA fragments
j. Mung bean nuclease: Suibstrate: Single stranded DNA, Products: released
deoxynucleotide monophosphates
k. Terminal deoxynucleotide transferase: Substrate: 3’ hydroxyl of DNA &
dNTPs; Products: DNA chain extended on 3’ end by one or more nonencoded dNMPs
6) (5 pts) From lectures. Outline all steps needed to complete an RNase-protection
assay to compare the mRNA abundance of the actin transcript in liver and heart
tissue. Briefly, a synthetic antisense RNA is prepared (e.g., using a cloned copy
of the gene in a vector like pTZ18u) and radiolabeled either during synthesis (like
we did) or afterwards with T4 polynucleotide kinase. The labeled RNA is
designed to be slightly longer that the natural transcript. The labeled RNA
annealed to the mRNA population from the tissue of interest and subsequently
treated with a single-stranded ribonuclease such as S1, T1 or RNase A. The
products are then resolved by gel electrophoresis. The RNase destroys any
unannealed probe and the resulting processed band intensities reflects the
relative abundance of the mRNA in the two tissue preparations.
7) (12 pts) From last year’s exam, lab activities and reading assignments. Given
the following hybridization data, determine the location and direction of
transcription for each RNA transcript. Assume that no more than one transcript is
present in the interval defined by two adjacent restriction sites (that is, EcoRI to
HindIII, HindIII to BamHI, BamHI to KpnI) and that no transcripts extend into an
adjacent fragment. Put an arrow head at the 3’ end of the transcript (5’ -> 3’)
and be sure to indicate clearly by a solid line labeled with a transcript length
where each transcript resides and its size.
Single stranded Probe (5’->3’) Hybridizing Bands on Blot (all in kb)
EcoRI –> HindIII
0
BamHI - >KpnI
1.4
KpnI ->EcoRI
0.79, 1.8
KpnI ->HindIII
0.79
EcoRI
5’X-------------
1.8 kb
HindIII
X------------------------
0.79
BamHI
KpnI
X------------------------X 3’
1.4
8) (2 pts) From lab activities. Which of the following steps is expected to increase
the number of E. coli transformants of pTZ19u (circle all that apply)
- Heat shock of the DNA + E.coli at 42C for 1.5 minutes
- Addition of DMSO to the competent cell preparation
9) (2 pts) From lab activities. Which of the following steps is expected to increase
the number of yeast transformants of YCplac33 (circle all that apply)
- Addition of excess denatured salmon sperm DNA during the
transformation
- Washing the yeast culture with lithium acetate (LiOAc) prior to DNA
addition
10) From lab activities. We prepared ssDNA for the recombinant pTZ18u and
pTZ19u transformants and ran a gel containing the following lanes: 1) uncut
recombinant pTZ18u plasmid isolated from E. coli, 2) single-stranded
recombinant pTZ18u, 3) single-stranded recombinant pTZ19u, 4) the annealed
mix of single-stranded recombinant pTZ18u and pTZ19u prepared in class.
a.
(3 pts)Add bands to this mock
gel that illustrate the relative mobilities of each form of DNA. Assume that the
wells are on top and for lane 1 be sure to label the relative mobility of the
supercoiled plasmid DNA, open circle (relaxed) plasmid DNA, and linear plasmid
DNA.
b. (2 pts) Draw a free-hand image of the predicted configuration of the DNA in lane
4. That is, assume that you could directly see the shape of the DNA, for instance
by using an electron microscope. What would it look like?
11) From lectures, lab activities and reading assignments. When using C. elegans
in an RNAi experiment, we simply added E. coli to the nematode.
a. (5 pts) How did the E. coli addition direct degradation of specific target
mRNAs (e.g., CLF1, DPY-1 etc) within the animal? Each E. coli strain was
transformed with a plasmid that included the target gene flanked by T7 RNA
polymerase promoters. The E. coli was plated on medium containing IPTG to
induce the expression of the T7 RNA polymerase gene which transcribed the
target gene and produced dsRNA. The bacteria were fed to the C. elegans
nematode where the microbially produced dsRNA was cleaved by DICER to
~22-24 nt segments. One strand of this RNA was then loaded onto RISC to
target the corresponding endogenous mRNAs for destruction.
b. (3 pts) Unlike most RNAi approaches, the RNAi affect is passed on though
generations of animal. That is, the offspring of treated animals still show
knockdown of the targeted transcript. An RNA-dependent RNA polymerase
is specifically responsible for this persistence across generations. Discuss
how this might occur. C. elegans has a potent RNA-dependent RNA
polymerase which is capable of using the “DICED” RNA from the initial
infection as primers to synthesize the equivalent of full length antisense mRNA
from the corresponding endogenous transcripts. These are passed on through
the sex cells to continue the dsRNA triggered RNA interference.
12) (2.5 pts) From lab activities. Two students cultured recombinant pTZ19u for
single stranded DNA production. Both students grew the cells properly and used
identical amounts of all materials (e.g., cells, virus, reagents etc) except that
student #1 failed to add kanamycin sulfate to the growing cells. While both
cultures grew well, the yield of ssDNA from student culture #1 was only 1% of
that for student culture #2. Offer an explanation. The M13K07 virus encodes the
kanamycin resistance gene. If no kanamycin is added, uninfected cells continue
to grow but since these lack the M13K07 DNA polymerase no ssDNA is made
(lowering student #1’s yield).
13) From lab activities. The melting curve of a student’s real-time PCR experiment
showed 2 peaks rather than one.
a. (2 pts) Offer an explanation for this result. Two different PCR fragments
were generated.
b. (2 pts) Based on the threshold value, the student’s realtime PCR results
suggest a concentration of 1.3 µg of viral DNA per ml of blood. Based on
part “a” of this question, what caution do you offer this student concerning
interpretation of her results? The data likely overestimate the viral load
since the second PCR fragment (which contributes to the “threshold
value”) may have no relationship to the virus but is included in the
measurement.