Rlegaspi Week 4

Transcription and Translation “Taken to the Next Level”

This computer exercise examines gene expression at a much more detailed level than before, requiring knowledge in both the biological aspects of the process and the translation of these steps into computer text-processing equivalents.

To begin the assignment, I needed to log into my account through the Terminal application on my MacBook:

ssh rlegaspi@my.cs.lmu.edu

And I typed in my password; then, I accessed the Dondi's folder which contained the infA-E.coli-K12.txt file:

cd ~dondi/xmlpipedb/data 

To view the specific sequence that we are practicing for this week's assignment I inputed the following command:

cat infA-E.coli-K12.txt
ttttcaccacaagaatgaatgttttcggcacatttctccccagagtgttataattgcggtcgcagagttggttacgc
tcattaccccgctgccgataaggaatttttcgcgtcaggtaacgcccatcgtttatctcaccgctcccttatacgtt
gcgcttttggtgcggcttagccgtgtgttttcggagtaatgtgccgaacctgtttgttgcgatttagcgcgcaaatc
tttacttatttacagaacttcggcattatcttgccggttcaaattacggtagtgataccccagaggattagatggcc
aaagaagacaatattgaaatgcaaggtaccgttcttgaaacgttgcctaataccatgttccgcgtagagttagaaaa
cggtcacgtggttactgcacacatctccggtaaaatgcgcaaaaactacatccgcatcctgacgggcgacaaagtga
ctgttgaactgaccccgtacgacctgagcaaaggccgcattgtcttccgtagtcgctgattgttttaccgcctgatg
ggcgaagagaaagaacgagtaaaaggtcggtttaaccggcctttttattttat

Modifying the gene sequence string: Highlighting ("Tagging") the special sequences within the gene

Completing this assignment required a review of Introduction to the Command Line page and a reading of the More Text Processing Features page. In class, my Homework Partner Anu Varshneya and I were able to get clues and hints on how to complete our assignment from Professor User:Dondi.

-35 box and -10 box

Firstly, we were able to learn the placement of possible -35 box and -10 box locations in the sequence and how to "tag" these special sequences. We were able to search for both of the special sequences concurrently with the knowledge that the sequence for a -35 box is tt[gt]ac[at] and the sequence for a -10 box is [ct]at[at]at. We could have used grep to search for the sequences individually, but to see where the potential locations for both -35 box and -10 box we used the following command:

cat infA-E.coli-K12.txt | sed "s/tt[gt]ac[at]/ *&* /g" | sed "s/[ct]at[at]at/ **&** /g"
ttttcaccacaagaatgaatgttttcggcacatttctccccagagtgt **tataat** tgcggtcgcagagttggttacgct
cattaccccgctgccgataaggaatttttcgcgtcaggtaacgcccatcgtttatctcaccgctcccttatacgtt
gcgcttttggtgcggcttagccgtgtgttttcggagtaatgtgccgaacctgtttgttgcgatttagcgcgcaaatc
*tttact* ta *tttaca* gaacttcgg **cattat** cttgccggttcaaattacggtagtgataccccagaggattagatggcc
aaagaagacaatattgaaatgcaaggtaccgttcttgaaacgttgcctaataccatgttccgcgtagagttagaaaa
cggtcacgtggttactgcacacatctccggtaaaatgcgcaaaaactacatccgcatcctgacgggcgacaaagtga
ctgttgaactgaccccgtacgacctgagcaaaggccgcattgtcttccgtagtcgctgattgttttaccgcctgatg
ggcgaagagaaagaacgagtaaaaggtcggtttaaccggcctttttattttat

After finding the locations of the potential -35 box and -10 box locations, it was clear where the actual -35 box and actual -10 box were (the first instance of the sequence for the -35 box and the second instance of the sequence for the -10 box). To ensure that we would only be highlighting the actual -35 box and -10 box sequences, all we need to do is replace the "g" in sed "s///g" with a "1" or "2" or the desired instance. This tells sed to replace the instance we desire, which is what was described in the More Text Processing Features page. The resulting command would produce the following result:

cat infA-E.coli-K12.txt | sed "s/tt[gt]ac[at]/ <minus35box>&<\/minus35box> /1" | 
sed "s/[ct]at[at]at/ <minus10box>&<\/minus10box> /2" 
ttttcaccacaagaatgaatgttttcggcacatttctccccagagtgttataattgcggtcgcagagttggttacgctcattacccc
gctgccgataaggaatttttcgcgtcaggtaacgcccatcgtttatctcaccgctcccttatacgttgcgcttttggtgcggcttag
ccgtgtgttttcggagtaatgtgccgaacctgtttgttgcgatttagcgcgcaaatc 
<minus35box>tttact</minus35box> tatttacagaacttcgg <minus10box>cattat</minus10box> 
cttgccggttcaaattacggtagtgataccccagaggattagatggccaaagaagacaatattgaaatgcaaggtaccgttctt
gaaacgttgcctaataccatgttccgcgtagagttagaaaacggtcacgtggttactgcacacatctccggtaaaatgcgcaa
aaactacatccgcatcctgacgggcgacaaagtgactgttgaactgaccccgtacgacctgagcaaaggccgcattgtcttc
cgtagtcgctgattgttttaccgcctgatgggcgaagagaaagaacgagtaaaaggtcggtttaaccggcctttttattttat

Transcription Start Site (TSS)

After finding and tagging the -35 and -10 box, the assignment required us to highlight the transcription start site (TSS), which is the twelfth nucleotide after the first nucleotide in the -10 box. Therefore, I could infer that the character 7 nucleotides after the end of the -10 box tag is the TSS because the -10 box sequence is 6 nucleotides long. In order to find the TSS with command sequences, I will need to create a new line using a technique described in More Text Processing Features. With the technique, I will tag the TSS in line 2 which will be the first nucleotide on the line.

cat infA-E.coli-K12.txt | sed "s/tt[gt]ac[at]/ <minus35box>&<\/minus35box> /1" | 
sed "s/[ct]at[at]at/ <minus10box>&<\/minus10box> /2" | sed -r "s/<\/minus10box> (.)6/&\n/g" | 
sed "2s/^./ <tss>&<\/tss> /g"
ttttcaccacaagaatgaatgttttcggcacatttctccccagagtgttataattgcggtcgcagagttggttacgctcattaccccgctgccgataaggaatttttc
gcgtcaggtaacgcccatcgtttatctcaccgctcccttatacgttgcgcttttggtgcggcttagccgtgtgttttcggagtaatgtgccgaacctgtttgttgcga
tttagcgcgcaaatc <minus35box>tttact</minus35box> tatttacagaacttcgg <minus10box>cattat</minus10box> cttgcc
<tss>g<\tss> gttcaaattacggtagtgataccccagaggattagatggccaaagaagacaatattgaaatgcaaggtaccgttcttgaa
acgttgcctaataccatgttccgcgtagagttagaaaacggtcacgtggttactgcacacatctccggtaaaatgcgcaaaaactacatccgc
atcctgacgggcgacaaagtgactgttgaactgaccccgtacgacctgagcaaaggccgcattgtcttccgtagtcgctgattgttttaccgcc
tgatgggcgaagagaaagaacgagtaaaaggtcggtttaaccggcctttttattttat

Ribosome Binding Site (RBS)

Now that I've found the TSS, it's time to find the ribosome binding site (RBS) and tag the RBS. In order to find and tag the RBS, I used the same technique as the previous portion of the assignment (finding the TSS). The special sequence for the RBS is gagg as indicated in the hints section of the homework assignment.

cat infA-E.coli-K12.txt | sed "s/tt[gt]ac[at]/ <minus35box>&<\/minus35box> /1" | 
sed "s/[ct]at[at]at/ <minus10box>&<\/minus10box> /2" | sed -r "s/<\/minus10box> (.)6/&\n/g" | 
sed "2s/^./ <tss>&<\/tss> /g" | sed "s/<\/tss> /&\n/g" | sed "3s/gagg/ <rbs>&<\/rbs> /1"
ttttcaccacaagaatgaatgttttcggcacatttctccccagagtgttataattgcggtcgcagagttggttacgctcattaccccgctgccgataaggaatttttc
gcgtcaggtaacgcccatcgtttatctcaccgctcccttatacgttgcgcttttggtgcggcttagccgtgtgttttcggagtaatgtgccgaacctgtttgttgcga
tttagcgcgcaaatc <minus35box>tttact</minus35box> tatttacagaacttcgg <minus10box>cattat</minus10box> cttgcc
 <tss>g<\tss> 
gttcaaattacggtagtgatacccca <rbs>gagg</rbs> attagatggccaaagaagacaatattgaaatgcaaggtaccgttcttgaa
acgttgcctaataccatgttccgcgtagagttagaaaacggtcacgtggttactgcacacatctccggtaaaatgcgcaaaaactacatccgc
atcctgacgggcgacaaagtgactgttgaactgaccccgtacgacctgagcaaaggccgcattgtcttccgtagtcgctgattgttttaccgcc
tgatgggcgaagagaaagaacgagtaaaaggtcggtttaaccggcctttttattttat

Start Codon

Finding the start codon is the same process as the previous two highlights: Add a new line after the tag (in this case, the RBS) and search for the special sequence (in DNA, it would be ATG because in mRNA it is AUG that codes for methionine [M] or the start codon).

cat infA-E.coli-K12.txt | sed "s/tt[gt]ac[at]/ <minus35box>&<\/minus35box> /1" | 
sed "s/[ct]at[at]at/ <minus10box>&<\/minus10box> /2" | sed -r "s/<\/minus10box> (.)6/&\n/g" | 
sed "2s/^./ <tss>&<\/tss> /g" | sed "s/<\/tss> /&\n/g" | sed "3s/gagg/ <rbs>&<\/rbs> /1" | 
sed "s/<\/rbs> /&\n/g" | sed "4s/atg/ <start_codon>&<\/start_codon> /1"
ttttcaccacaagaatgaatgttttcggcacatttctccccagagtgttataattgcggtcgcagagttggttacgctcattaccccgctgccgataaggaatttttc
gcgtcaggtaacgcccatcgtttatctcaccgctcccttatacgttgcgcttttggtgcggcttagccgtgtgttttcggagtaatgtgccgaacctgtttgttgcga
tttagcgcgcaaatc <minus35box>tttact</minus35box> tatttacagaacttcgg <minus10box>cattat</minus10box> cttgcc
 <tss>g<\tss> 
gttcaaattacggtagtgatacccca <rbs>gagg</rbs> 
attag <start_codon>atg<\/start_codon>  gccaaagaagacaatattgaaatgcaaggtaccgttcttgaa
acgttgcctaataccatgttccgcgtagagttagaaaacggtcacgtggttactgcacacatctccggtaaaatgcgcaaaaactacatccgc
atcctgacgggcgacaaagtgactgttgaactgaccccgtacgacctgagcaaaggccgcattgtcttccgtagtcgctgattgttttaccgcc
tgatgggcgaagagaaagaacgagtaaaaggtcggtttaaccggcctttttattttat

Stop Codon

Figuring out how to find the stop codon in the DNA sequence because I had to account for the fact that the stop codon was a multiple of 3 codons away from the start codon; in addition, the stop codon must have the special sequences of either TAA, TAG, or TGA (in mRNA it would read as UAA, UAG, and UGA) and the stop codon has to come before the terminator of the DNA sequence. With all of those factors in mind, the locating and tagging of the stop codon required a more complex sed command. Firstly, I created a new line after the start codon sequence. Afterwards, I needed to use the more complex sed command (use of sed -r "..."). I spaced the nucleotides that occurred after the start codon into groups of three, then I needed to identify the first instance of a stop codon and tag it. To bring the sequence back together (without spaces), I provided a command for that and re-labeled the tags. The command sequence used and results were as follows:

cat infA-E.coli-K12.txt | sed "s/tt[gt]ac[at]/ <minus35box>&<\/minus35box> /1" | 
sed "s/[ct]at[at]at/ <minus10box>&<\/minus10box> /2" | sed -r "s/<\/minus10box> (.)6/&\n/g" | 
sed "2s/^./ <tss>&<\/tss> /g" | sed "s/<\/tss> /&\n/g" | sed "3s/gagg/ <rbs>&<\/rbs> /1" | 
sed "s/<\/rbs> /&\n/g" | sed "4s/atg/ <start_codon>&<\/start_codon> /1" | 
sed "s/<\/start_codon>/&\n/g" | 
sed -r "5s/.../& /g;5s/taa|tag|tga/ <stop_codon>&<\/stop_codon> /1;5s/   //g;5s/<stop_codon>/&/g;5s/<\/stop_codon>/& /g"
ttttcaccacaagaatgaatgttttcggcacatttctccccagagtgttataattgcggtcgcagagttggttacgctcattaccccgctgccgataaggaatttttc
gcgtcaggtaacgcccatcgtttatctcaccgctcccttatacgttgcgcttttggtgcggcttagccgtgtgttttcggagtaatgtgccgaacctgtttgttgcga
tttagcgcgcaaatc <minus35box>tttact</minus35box> tatttacagaacttcgg <minus10box>cattat</minus10box> cttgcc
 <tss>g<\tss> 
gttcaaattacggtagtgatacccca <rbs>gagg</rbs> 
attag <start_codon>atg<\/start_codon>  
gccaaagaagacaatattgaaatgcaaggtaccgttcttgaa
acgttgcctaataccatgttccgcgtagagttagaaaacggtcacgtggttactgcacacatctccggtaaaatgcgcaaaaactacatccgc
atcctgacgggcgacaaagtgactgttgaactgaccccgtacgacctgagcaaaggccgcattgtcttccgtagtcgc <stop_codon>tga</stop_codon> 
ttgttttaccgcctgatgggcgaagagaaagaacgagtaaaaggtcggtttaaccggcctttttattttat

Terminator

Finding and tagging the terminator was one of the easier tasks of this assignment because of the hints given on the assignment description page, in-class work, and the continuous practice of sed on this assignment.

Electronic Lab Notebook

• Discussion in class with Anu - Finding the minus35box and Finding the minus10box and inserting the descriptions around the codes (in-class work time) - To be written

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Ron Legaspi
BIOL 367, Fall 2015

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Homework Partner: Anu Varshneya