BISC411
Experimental Molecular Biology of the Cell
Questions to consider as you read the T4 lysozyme paper from the
Journal of Biotechnology that I gave to you:
- What is pHSe5?
- What is wt* ?
- What was the size of the T4 lysozyme gene?
- How was that determined?
- What is the difference between the wt* gene and the newly
constructed
mutant
gene?
- What is the size of the new mutant gene?
- What is pQR752?
- What differences are predicted if pHSe5 and pQR752 were run on an
agarose
gel uncut?
- Cut with Bam H1?
- Cut with Hind III?
- Cut with EcoR1?
- How can you distinguish pHSe5 from pQR752?
- What was used to induce the transcription of the T4 lysozyme gene
in
the
experiments in the paper?
- What is IPTG?
Questions to consider as you read the
CIB paper published in the Journal of Biological Chemistry that I sent
to you:
- What is clone 8?
- How many base-pairs are in clone 8?
- What size is the open reading frame from clone 8?
- What does the clone 8 sequence predict is the size of the CIB
protein it codes for?
- Clone 8 was subcloned into pGEX-2T which we are using in BISC411
this semester. It was cloned into the vector using Bam H1 and EcoR1
restriction sites. Using the information you have about clone 8 and
also the pGEX-2T map you were sent, predict the sizes of DNA fragments
in base pairs you would see on an agarose gel following digestion of
this CIB expression plasmid with BamH1; with EcoR1; or with both BamHI
and EcoR1.
- What did the authors of this paper conclude about the CIB protein
produced from clone 8? Specifically, what chemical behavior
(hydrophilic or hydrophobic overall); what overall charge; what
isoelectric point.
- Does CIB associate with a membrane, and if so, how?
Questions to consider as you read the CIB protein structure paper
published in the journal Protein Science that I sent to you:
- How many amino acids per chain were identified by x-ray
crystallography?
- The protein crystallized as a dimer, is this physiologically
accurate?
- What amino acids of CIB, deduced by the sequence of clone 8 (see
above) are not represented in the crystal structure?
- What likely CIB functional domain is missing due to this?
- Why was there a molecule of GSH present on one chain of the dimer
in the crystal?
Questions to consider as you examine the sequence and other documents
related to Sprinter that I sent to you:
- The plasmid you will be working with contains a cDNA that codes
for only a portion of the Sprinter protein (amino acids 40-240). It is
cloned into the expression vector p-MAL. You have been given the
restriction enzyme map and recognition sequences of the p-MAL vector
that was used during the subcloning of the Sprinter segment cDNA.You
have also been given the restriction enzyme map of the recombinant
plasmid that contains the cDNA, showing all ot the unique restriction
enzyme cutting site sites within it. Finally, you have also been given
a complete DNA sequence of the recombinant plasmid. Use these to answer
the questions below.
- Do you see the ECOR1 cutting sequence (GGATTC) in the
recombinant plasmid sequence? Do you see a HindIII cutting site
(CAAGCTT)?
- What is the distance between the EcoR1 and HindIII sites
according to the original, unaltered vector? What is the distance
between these sites in the recombinant plasmid according to the
restriction enzyme map?
- According to the DNA sequence of the recombinant plasmid,
the inserted Sprinter
segment cDNA is 629 basepairs in size (count it and see if you agree).
During the subcloning procedure, for a variety of reasons, the plasmid
was altered by including another enzyme recognition sequence, that of
SpeI (ACTAGC). Do you see that cut site in your sequence? A few
other changes also happened.This accounts for the difference in size
between the two EcoR1 to HindIII distances.
- Using this information, predict the sizes of DNA fragments you
would
expect to see on an agarose gel following digestion with EcoR1; with
HindIII, and with EcoR1 and HindIII. We are using
the pMAL-c4G vector which is 6646 basepairs in
size before any manipulations or insertions.