Biochemistry

• (60 pts) Arabinose Operon Gene Expression
  1. (5 pts each for activator and repressor description)Arabinose is an alternative food source (similar to lactose in coli). The operon and its regulation is picture right.The araC regulator works as both an activator and repressor.  Describe how this works (this might take some online research).
  2. (5 pts for each condition listed)Describe the 4 conditions and what would be bound to the gene if
     1. Glucose is high and arabinose is high
     2. Glucose is high and arabinose is low

• Glucose is low and arabinose is high

1. Glucose is low and arabinose is low

1. (5 pts for each induction listed with explanation) This regulation system is often used for control of recombinant plasmids (referred to as pBAD). Describe how you might use pBAD with the pictured pET vector below to induce expression of 2 recombinant genes at different timepoints.  (Induce the pET vector first then pBAD).
2. (5 pts for explanation) Why are these plasmids compatible (assuming different origins)?
3. (5 pts for enzyme pair and 2.5 pts correct orientation on each plasmid) Pick 2 restriction enzymes for each plasmid to insert a gene of interest. Make sure they are in the correct orientation for expression (you can use the same one for both plasmids if desired/possible).

• (25 pts) Reference the attached paper for the following question. Note that you do not need to understand all details of this paper to answer the questions, you may also consult other sources.  I understand this paper is a bit more advanced so please ask if you have questions.  I am more discussing with these questions general approaches to unnatural amino acid incorporation.
  • (5 pts explanation) Explain why this method is called nonsense suppression…how does it work?
  • (10 pts explanation) Explain (in terms of translation) why removing RF1 would increase the amount of unnatural amino acid added to proteins possessing the UAG mutated gene sequence?

2. (10 pts explanation)Looking at figure 2d) pictured right. What do you think the lower band is in lanes 2-3 for 1-TAG expression with JX2.0? Why is this occurring? Note: We know that the JX2.0 strain has RF1 still while JX3 is missing RF1. 

• (35 point) Since COVID has started, you have become quick bored and decided to do some at home science. You randomly start swabbing things in your home to see what of fungi kind grows….to your amazement (and disgust) you discover a new type of fungi!  You want to find out the genomic sequence for it. 
  1. (5 points)What method do you use and why?
  2. (10 points)You get the results back see a region you are particularly interested in and decided to resequence with Sanger sequencing to confirm your results. You are old school and run it on a gel.  What would the following sequence look like on the following gel template below: 5’ – atgctcacgt – 3’ (you may draw on this and add the picture or hand draw your own)
  3. (5 points explanation, 5 points new lane image) What would happen if the ddGTP was contaminated with ddTTP? What would lane 3 look like instead?
  4. (10 points) If you wanted to look at the expression of a gene in your new organism, describe a method you could use to quantify mRNA concentrations.

• (25 points) Based on the figure below,(5 pts)1) explain why this level of selectivity is necessary for tRNA synthetase loading (in terms of protein production accuracy), (10 pts)2) explain where different enzymatic activities are occurring in the tRNA synthetase,(10 pts)3) explain how this 2 sieve system might work to differentiate between isoleucine and valine.

Structural Basis for Double-Sieve Discrimination of L-Valine from L-Isoleucine and L-Threonine by the Complex of tRNAVal and Valyl-tRNA Synthetase, Cell, Volume 103, Issue 5, 2000, Pages 793-803

• (10 pts explanation) Give 2 different forces/interactions that give stability to the DNA double helix structure.

• (5 pts explanation) Explain how acetylation and methylation of histones changes gene expression.

• (40 pts)
  1. What is your disease?
  2. (5 pts)Find a paper that discusses a genetic study done on your disease (this could be anything from cloning and expressing a protein to DNA microarrays etc….tons of options here).
  3. (5 pts)What was the genetic study method used?
  4. (10 pts, detail and accuracy) Explain how they accomplished the experiment listed in part c).
  5. (10 pts) What did you learn from this study about your disease? What does it contribute to the field of knowledge?
  6. (10 pts) Propose a genetic experiment for your disease based on the research you have read about so far between Exams 1 and 2. What would you accomplish with this study and why is it useful?