MCB 421
Homework 11
Due Nov 29
Fall 2011
The trpA gene is required for the biosynthesis of the amino acid tryptophan. trpA
mutants require tryptophan for growth. You have made a derivative of a pir-dependent
vector that has two fragments that code for the beginning and end of the trpA gene fused
to each other as shown in the figure below. The vector also carries a gene that makes
cells resistant to tetracycline (tetA) and a gene (sacB) that makes cells sensitive to 5 %
sucrose. Expression of both tetA and sacB is driven by promoters on the plasmid and are
expressed in cells.
1.
a). Describe how you could select for integration of this plasmid into the chromosome of
a trpA+ cell. Be sure to draw clearly the genetic event(s) that are required.
ANSWER: Plate on Minimal Media + Tetracycline.
tetA+
sacB+
ori R6K
0
0
1600
200
tetA+
1500
1600
sacB+
ori R6K
TrpA+ TetR SacS
trpA+
MCB 421
Homework 11
Due Nov 29
Fall 2011
b. Would the resulting strain be Trp+ or Trp- ? Be sure your drawing supports your
answer.
ANSWER: Trp+
c. How could you select for cells that have lost the plasmid?
MM + Trp –Tc + 5% Sucrose.
d. Draw out the genetic events that could occur during loss of the plasmid. Indicate the
structure of the trpA gene left behind. (Hint: There are two different ways the plasmid
can excise). What would be the tryptophan phenotype of each type?
sacB+
tetA+
sacB+
trpA+
tetA+
trpA+
ori R6K
ori R6K
trpA+
trpA-
TrpA+ TetS SacR
TrpA- TetS SacR
2. The adhE gene is about 2000 bp. Nancy Maulen cloned a fragment corresponding to
nucleotide 50 to 500 of the S. typhimurium adhE gene into the pir-dependent plasmid
pGP704. [pGP704 has no homology with the S. typhimurium chromosome.]
amp
oriR6K
'adhE'
50
500
a.
Why is this plasmid pir-dependent?
ANSWER: The pir gene encodes a protein required for plasmid replication.
MCB 421
b.
Homework 11
Due Nov 29
Fall 2011
What would happen if this plasmid was transformed into a S. typhimurium adhE+
R
pir+ strain with selection for Amp ?
ANSWER: The plasmid would replicate independently.
c.
What is the predicted phenotype of the transformants described in part b above?
Why?
ANSWER: AdhE+ AmpR
The plasmid would not disrupt the chromosomal adhE gene.
d.
What would happen if this plasmid was transformed into a S. typhimurium adhE+
R
pir- strain with selection for Amp ?
ANSWER: The plasmid would integrate into the chromosome by homologous
recombination via the adhE homology.
e.
What would the phenotype of the transformants described in part d above? Draw
a diagram to explain your answer.
ANSWER: AdhE- AmpR
3. You recently discovered a new plasmid from an environmental isolate of E. coli B, which you
named pCar33. It carries resistance to ampicillin.
a. What DNA sequence would be required for the plasmid to transfer by conjugation? What class
of enzymes mediates this process?
Answer: It would require an oriT. To start the transfer process, a tyrosine recombinases
nicks the oriT and starts transfer. Other proteins involved are helicases to pump the DNA,
misc enzymes to form relaxasome.
b. You attempt to mate pCar33 into a closely related species of E. coli B-12, a strain of
Salmonella 215, and a strain of Bacteroides theta. After mating, you only find colonies when
pCar33 was mated into the E. coli B -12 recipient. What can you conclude about its host range?
Why was this experiment not conclusive?
Answer: It has the ability to mate and replicate in E. coli B-12. It may not be able to mate
or replicate in the other two strains. You cannot assume it lacks the ability to mate.
c. Dr. Farrand discussed the only two accepted by him experimental ways to definitively
determine host range of a plasmid during his lecture. Choose one of the ways and diagram how
you would set up the experiment of mating pCar33 into E. coli B-12, a strain of Salmonella 215,
and a strain of Bacteroides theta. Include cell types, media, and a diagram of all constructs you
might use.
1. Transform into the E. coli B strain a mobilizable plasmid with a broad host range such
as RP4. Mate the cells to each of the recipients and plate on selective media (can be
determined by the student). If pCar33 is capable of mating and transferring DNA into the
MCB 421
Homework 11
Due Nov 29
Fall 2011
recipient, RP4 will co-mobilize. After the mating is complete RP4 could be detected by
plating on selective media or isolation.
2. Insert a transposon on pCar33. If the plasmid can conjugated and transfer DNA into the
recipient, the transposon can also move into the recipient. Once it is insde the cell, it can
transpose into the chromosome for example and be detected by selectable media.
d. Finally, during one of your matings of pCar33 into a new strain of E. coli B-17, very few
colonies are recovered <10-8. After careful thought, you realize you may have a case of zygotic
induction. What does this mean?
Answer: Your strain of E. coli B containing pCar33 must have been a lambda lysogen.
The new strain of E. coli B-17 must not have lambda. When pCar33 transfers into B-17, no
C1 repressor is there to force lambda into a lysogenic lifecycle. Most of the time, the virus
goes lytic and kills the cells. However, it is possible to also choose lysogenic at a low
frequency and form lysogens again.
4. a. Plasmids often have oriT and oriV sites. What is the function of each?
oriT sites are sites used for nicking of DNA in preperation for DNA transfer to a recipient.
oriV sites are used for initiation of bidirectional DNA replication.
b. Do the sites function in cis or in trans (or both)?
oriT sites can function in both cis and trans. The site is the important for nickase protein
recognition. OriV sites only work in cis because they are the sites for initiation of DNA
synthesis.
c. E. coli mutants that have a temperature sensitive mutation in the dnaA gene (dnaATS) can
initiate chromosomal DNA synthesis at 30o C but not at 42o C. Thus cells are inviable at 42o C.
If one grows cells containing a dnaATS mutation and the F plasmid at 42o C, some rare colonies
grow. What event likely accounts for this observation? What is an obvious genetic prediction
from your answer?
Most likely F has integrated into the host chromosome to form an Hfr. The host
chromosome is replicated by initiation of DNA synthesis at the oriV site. The Hfr should
transfer chromosomal genes to an F- strain in a conjugation experiment. This is a real
technique for isolation of Hfs called integrative suppression.
5. Microarrays, proteomics and RTq-PCR are techniques for measuring various aspects of
gene expression. For each technique, describe the application with respect to measuring
A). Transcription regulation
B). Translation regulation
C). Post- translational modification of a protein.
Answers:
MCB 421
Homework 11
Due Nov 29
Fall 2011
A). Microarrays and RTq-PCR can be used to measure transcription because they
measure RNA levels. Proteomics only measure the amount of protein made so
effects at the transcriptional and translation levels can affect the end result.
B). None of the methods directly measures translational regulation.
C). None of the methods directly detects post-translational modification of a protein.