Practical Application #4 Instructions and Worksheet_2022(1) (1)

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Feb 20, 2024

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Practical Application #4 Name: Section: Group Number: For this assignment, you will be learning how to search for gene sequences on NCBI, and how to use the online B asic L ocal A lignment S earch T ool (BLAST). For the context of this tutorial we will be interested in the LuxI gene sequence and comparing between different organisms. 1. For review, what protein product does LuxI encode for? What does this protein (and any enzymatic product of this protein) do? 2. Outside of Allivibrio fischeri , what other organism(s), either species or genera, would you anticipate homologous LuxI sequences to be present in? Why? Before we can BLAST, the reference sequence is needed. Go to the NCBI website ( https://www.ncbi.nlm.nih.gov/ ) and under the dropdown menu that says “All Databases” select “Gene.” In the text box, type “LuxI” and hit search. Select the first gene result, the autoinducer synthesis protein for “ Vibrio [sic] fischeri .” You may also use a different search result for an autoinducer synthesis protein if you like, for additional practice. Frequently you will find unusual genetic associations between organisms you would not have anticipated! 3. Why would a similar gene sequence be seen in two organisms that are not closely related?

Scroll down to “Genomic regions, transcripts, and products.” This is a convenient genome browser that lets you visualize the organism’s genome, and provides useful links. Currently it is centered on your autoinducer of choice, however you can zoom out or drag the window to see neighboring regions of the genome. Next click FASTA (pronounced “Fast Ayyyyyyy” *thumbs up*). This directs you to the gene sequence in FASTA format that can be used in a variety of ways, including many offline tools. For now, simply copy and paste the sequence into a text pad (or leave this window open and proceed in a new tab). BLASTN In the new tab, go to the NCBI website ( https://www.ncbi.nlm.nih.gov/ ) and then click “BLAST” on the right-hand side. Notice that there are several ways of performing a BLAST search, and we will be covering two of them. 4. While we won’t cover the reverse situation of performing a BLAST search when starting with a protein sequence, describe a potential scenario when you would have a protein sequence, but want to BLAST for a nucleotide sequence. Click nucleotide BLAST (nucleotide -> nucleotide) button. This type of search expects a nucleotide reference sequence, and looks for similar nucleotide sequences in the database. Input your sequence for the luxI gene. Sometimes BLAST searches can return a lot of unwanted results. Since luxI is frequently used in plasmids, many cloning vectors are going to be listed, however for the context of this assignment we’re not interested in those. Under “Choose Search Set,” “Organism,” type in “taxid:29278” and from the list select “cloning vectors.” Then tick the “exclude” checkbox. This will remove them from the BLAST search. Under “Optimize,” click “somewhat similar sequences.” Just ‘cuz. 5. Before searching, what do you predict to be the main BLAST result? Why? Click BLAST, and wait for results. Sometimes this can take several minutes, and certain times of the day during high traffic can also affect this.

6. What is the best result (Species, E-value, coverage)? 7. Are there any other interesting results? (You may have to scroll down a bit.) Write some here. 8. Which of these would you expect? Why? Scroll down past “Descriptions” and look at “Alignments.” This is where each individual result from the list in “Descriptions” is shown in a visual alignment. The top line is the reference gene used to query the database, while the bottom line is what it was found to match up with. BLASTX Now return to the root BLAST page and this time click blastx (translated nucleotide -> protein). 9. What are the advantages of performing a BLAST based on the amino acid sequence encoded by the nucleotide sequence? Again paste your gene sequence into the query field. It will automatically be converted to an amino acid sequence by the program. Keep in mind that there are situations where the genetic code will be different, such as mitochondria, however for our purposes the standard genetic code is correct. Make sure to filter out cloning vectors from our search by typing in “taxid:29278” and clicking on “cloning vectors” and checking the exclude checkbox. 10. What is the best candidate gene and organism that BLAST returns now? 11. Note other BLAST hits in the results and compare with those alternate hits seen in the nucleotide – nucleotide BLAST from earlier. Have any shifted up in score? Down?

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12. Other than similarities between two different amino acid’s structures, give a reason for the shift in score. 13. As suggested in 12, if two different amino acids are “similar” enough, then sometimes a substitution won’t affect protein function. Scroll through the alignments and list three commonly seen substitutions (illustrated with a “+” symbol between the query and subject line), and give a reason those two amino acids might be interchangeable. 14. Do you see any high scores that are listed as “hypothetical protein?” Is this BLAST search sufficient to claim that those hypothetical proteins are indeed a functional luxI homolog? Why or why not? Include the species name associated with the hypothetical protein. 15. If you wanted to verify the function of one of these hypothetical proteins, what would be the next step? BONUS: List one interesting result you saw during your searches, and why it was interesting to you.

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