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?

• 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?

• 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?

• 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.