Michael R. Pina's Lab Notebook

Lab Notebook

Week 2

a) At around base 80, the upper (blue) DNA sequence has AC whereas the lower (yellow) DNA sequence has GG in the last exon.

b) No, they don't all have to have the same DNA sequence, they just don't have any of the correct pigment sequences or they do not have a promoter region.

c) All of them share the first two exon sequences, labeled in purple and teal; it is in the third exon sequence where they differ. This region is likely responsible for the different colors.

In order to create a blue flower to cross with the red flower, I loaded the Green-2 flower and arbitrarily changed some of the bases in the yellow sequence so that the new folded protein was white, creating a blue flower.

When I crossed my new blue flower with the pre-existing red flower, I got some purple but it was not true breeding. After some thought, I went back to both the blue and red flowers and observed the differences in their upper and lower sequences in order to change both of the DNA sequences so that they were true breeding red and blue, respectively. Crossing these 2 true breeding red and blue flowers yielded all purple flowers.

I was not able to isolate a purple flower that would produce all purple flowers when self-crossed so I began to observe the biochemistry and noticed that Tyr and Phe were the determinates for color in the flowers where Tyr produced blue and Phe produced red. Using this information, I began to play around with the DNA sequence, deleting, adding, or changing certain base pairs (around 80) to see the outcome. Finally, I thought to add both Tyr and Phe together which yielded a mutant purple flower. Mutating both the upper and lower DNA sequences allowed for a true-breeding purple flower.

Week 3

In class activity

What I know about HIV so far

HIV is a human pathogen that targets the immune system. It uses white blood cells as hosts; the white blood cells burst after being infected and thusly the immune system is compromised. The HIV virus itself does not actually kill anyone, rather people who otherwise become sick while they have the virus cannot fight off the disease (such as influenza). The virus is transmitted through the exchange of human tissue such as blood or other bodily fluids. When the number of HIV virus cells go beyond a certain percentage in humans, it is referred to as AIDS. There is no known cure for the virus as of yet, just some treatments that don't always work.

What I would like to know

1. What is it about the virus that makes it so difficult to treat?
2. How did the virus evolve to attack the immune system of its host?
3. What is the most recent advancement that has been made in hope of finding a cure?

Becoming an instant expert with PubMed

• Query functions much like Google.
• Click on a search result to get an abstract. The book mentions something about clicking on an author but that feature doesn't seem to be available any longer. The abstract can be saved in a number of ways by clicking the "Send To" link.
• Alternatively, you can choose one or more articles in a given page by selecting the check-mark boxes next to them. You can still use the "Send To" feature even for multiple articles.
• After clicking on an article you can choose to show the abstract, summary, etc. by clicking on "Display Settings."
• At the top right corner there's a link that says "Full Text Article in PudMed". From here you can view the full article, save it in a PDF, etc.

• Queries can be restricted using certain commands like [TIAB] to search only with in the title or abstract.

HIV paper

1. Berkhout B and Paxton WA. HIV vaccine: it may take two to tango, but no party time yet. Retrovirology. 2009 Oct 9;6:88. DOI:10.1186/1742-4690-6-88 |

Covers a breakthrough in Thailand.

• Using Google Scholar didn't yield the same search results, though I could find the article if I searched for the title.
• ISI was similar, this is likely due to the fact that the article has only been cited once and isn't very popular.
• That Markham et al. article has currently been cited 52 times.

Week 6

The S value for subject 10 across all visits is 74.

The harmonic sum is 4.54

θ would be 16.3

Min difference is 1.14

Max difference is 19.95

S10 FASTA File

S10 Phylogenetic Tree

S10 Rooted Phylogenetic Tree

S10 Distance Matrix

Week 8

1. I went to the uniport website at http://www.uniprot.org/ and in the search field I entered "gp 120"
   • This yielded over 4000 pages of results
   • I selected the first result (accession number P04578, secondary accession number O09779), which had an amino acid length of 856
   • I found that this contains protein sequences from both gp120 and gp41
   • I downloaded the FASTA file
2. I went to the Biology workbench site
   • I selected nucleic tools
   • I selected the nucleotide sequence (285bp long) from Subject 1, Visit 1-1
   • I viewed the sequence and copied it to my clipboard
3. I navigated to the ORF finder at http://www.ncbi.nlm.nih.gov/projects/gorf/ and pasted my nucleotide sequence into it
   • I chose the ORF of +1 because it yielded the most amino acids
   • The length of the ORF was 75 aa
4. I compared the output results from the ORF finder from the FASTA file retrieved from UniProt
   • I selected a string of amino acids (about 15 aa long) and using the "Find" feature, tried to find that same sequence in the FASTA file
     • This did not work
   • I tried selecting aa sequences from other ORFs and tried to do the same thing but I still got the same results
5. I used the other ORF finder posted in the wiki
   • I found that the output of this website is a little bit easier to read than the other website shown in the For Dummies book
   • Many of the ORFs had stop codons but one did not, so I determined that this was probably the correct sequence:
   • E V V I R S E N F T N N A K I I I V Q L N E S V E I N C T R P N N N T R K S I H I G P G R A F Y T T G D I I G D I R Q A Y C N I S R A E W D N T L K Q I V I K L R E H F G N K T I V F N H S S
6. I figured I was not getting any matching results due to changes in the amino acid sequence
   • I imported the S1V1-1 aa sequence in to the biology workbench
   • I did the same thing for the FATSA file obtained from UniProt
   • I performed a multiple sequence alignment using CLUSTALW
7. What I found was the most of the sequence was fully conserved between the two sequences
   • There were about about a dozen discrepancies due to amino acid change or deletion/insertion)
   • Also of note; there were about a dozen amino acids that were not fully conserved conserved

EVVIRSVNFTDNAKTIIVQLNTSVEINCTRPN
EVVIRSENFTNNAKIIIVQLNESVEINCTRPN
****** ***:*** ****** **********

NNTRKRIRIQRGPGRAFVTIG-KIGNMRQAHCNISRAKWNNTLKQIASKL
NNTRKSIHI--GPGRAFYTTGDIIGDIRQAYCNISRAEWDNTLKQIVIKL
***** *:*  ****** * *  **::***:******:*:******. **

REQFGNNKTIIFKQSS
REHFG-NKTIVFNHSS
**:** ****:*::**

• Where * indicates full conservation and : indicates strong group conservation, but not necessarily full conservation
• The top corresponds to the FASTA file from UniProt
• The bottom strand corresponds to S1V1-1 from the Markham article

FATSA file

Week 9

Subject 10 and 4 Box

Subject 10 and 4 Phylogenetic Tree

An option for a newer article with structure. This shows V3 in complex with CD4.

http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=78166

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