tants 1 and 2 explain why changing structure and sequence at the same time makes it hard to interpret the functional effect of a mutation (aka what was the reason for designing mutant 3) (7 pts.).
- Choose a mutant (from the three designed here) that you would like to test experimentally. Provide a brief explanation for why you chose this mutation (6 pts.).
BLAst Problem Set
Define or describe the following terms as used in BLAST:
- E (Expect or Expectation value)
- Max Score
Find and report the following:
- The number of identities in the alignment between the query and the subject, Cyanothece sp. PCC7425
- The percent of coverage between the query and the subject, Sulfuricurvum kujiense DSM16944
- The number of gaps in the alignment between the query and the subject, Clostridium kluvyeri DSM555
- The Expect value in the alignment between the query and the subject, Exiguobacterium sibiricum 255-15, complete genome.
- The percent of maximum identity between the query and the subject, Geobacillus sp Y412MC52
Find and interpret the following:
- Find the E value for the alignment between the query and the subject, Bacillus subtilis subsp. spizizenii str. W23, complete genome. How do you specifically interpret this number? (What does this number mean?)
- Find the E value for the alignment between the query and the subject, Pseudomonas stutzeri DSM 4166. How do you specifically interpret this number? (What does this number mean?)
- For the alignment between the query and the subject, Paenibacillus sp. JDR-2, complete genome, there are fourteen gaps reported yet only five gap openings are shown in the alignment. Is this a mistake? Briefly explain.
General BLAST interpretation:
- The search using megablast gave far fewer matches than the search using blastn. Check the Algorithm Parameters for both searches. Could the difference in word length explain the difference in matches? Briefly explain.
- The alignment with the subject, Clostridium cellulovorans 743B complete genome, has a smaller % identity than the subject, Lysinibacillus sphaericus C3-41, complete genome. Yet, these two subjects have the same maximum score. Briefly explain why this is the case.
Protein Properties Worksheet
In the past 10 years, one of the greatest improvements in biochemistry took place in the field of bioinformatics. Search engines and prediction tools enable us to easily learn a lot about a protein or RNA of interest before we actually start working on a project. The availability of these online tools makes it much easier to tailor a project and assess its feasibility. This problem set introduces you to a few easy-to- use prediction tools. You will learn how to calculate the molecular weight, isoelectric point or amino acid composition of a protein, how to perform sequence alignments to assess sequence conservation, how to predict secondary and tertiary structure.
Follow the step-by-step instructions below and answer all questions
You will use the sequence of the ykkCD multidrug-resistance efflux pump. Efflux pumps are an important part of bacterial defense against antibiotics: they pump antibiotics out of the bacterial cell thereby rendering them ineffective in treating bacterial infections. The RNA sensor (ykkCD riboswitch) you perform mutagenesis on in the biochemistry lab turns on production of the ykkCD efflux pump.
The ykkCD efflux pump is a heterodimer pump meaning that it is made out of two different proteins: ykkC and ykkD. In the first part of the exercise you will perform a series of predictions using these sequences to get an idea about the physico-chemical and structural properties of this pump.
The sequences of the ykkC and ykkD pumps are listed below:
- Generate a sequence alignment between the two pump monomers (C and D) using this link: http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_clustalw.html. Make sure you copy/paste both sequences into the program as it is shown above (names and > included).
- a) Paste the sequence alignment!
- b) How similar are these two sequences (% conservation)?
- c) Is there any trend in the 