Sbb14-Karim Merchant

Karim R. Merchant 01:50, 7 February 2014 (EST)

Lab Notebook created

January 30th and February 4th Golden Gate Assembly

Golden Gate Assembly Demo

Combining 5 "parts" into 1 "device". Digestion and Ligation will happen in same step.

Colonies: Green (want). White (something mis-ligated/star activity). Red (if RFP wasn't chopped out during digestion).

Did Transformation, and plating.

Results looked good- plenty of green colonies. 2 white colonies, and a few red colonies as well.

February 6th

Karim R. Merchant

00:11, 18 February 2014 (EST)

Our group members were assigned. I'm in Group 4 with: Chris Coates, Kelvin Li, Christy Truong, and Tae Won Chung. Our project is to make a cohort of aminoacyl tRNA synthetases for a thermophilic organism.

Job for next class: Find suitable themophilic bacteria to obtain tRNA synthetase genes from. Ex/ Thermus Thermophilus. Find papers that cite that organism's aaRSs to be suitable in e. coli. Use Pubmed for genome searches, and use multiple sequence alignment to match up the genes for all 20 synthetases to the ones in e. coli.

February 13th

Karim R. Merchant 00:33, 18 February 2014 (EST)

We had a debate during class discussing which thermophilic organisms to use to obtain aaRS genes which could be compatible with e. coli bacteria. Ended up deciding on 2: Thermus thermophilus and Thermotoga maritima. The Professor assigned teams 1 and 2 to work on Thermus thermophilus and teams 3 and 4 to work on Thermotoga maritima. Also, teams 1 and 3 will work on cloning the following amino acids: A C D E F G H I K L. Teams 2 and 4 will clone: M N P Q R S T V W Y. Within my team, I was assigned to clone W (Trp) and Y (Tyr). Both of my amino acids are single polypeptides, so only 1 gene needs to be cloned (unlike PheRS).

Thermotoga maritima: ATCC# 27634D-5 and Accession # AE000512.

Our backbone plasmid will be pBAD myc/his A. It can be obtained from invitrogen. We are to insert genes between NcoI and EcoRI. Designing primers: need 20 bp of perfect homology, and then add restriction sites/ extra bp at termini.

Process:

PCR

Gel cleanup

Digest PCR insert and plasmid (also phosphorylate)

Cleanup

Ligate

Transform

Rescue

Plate

February 19th

Karim R. Merchant 00:11, 20 February 2014 (EST)

I planned my construction files to insert TyrRS and TrpRS Thermotoga Maritima genes into the pBADmychisA plasmid. The TyrRS gene was slightly convoluted as it contained a NcoI internal restriction site. I used SOEing to get rid of it. My construction files are below.

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Construction file for TrpRS

PCR TrpRS-Fwd/TrpRS-Rv on Thermotoga Maritima genome  (1008 bp, pcrpdt)

Digest pcrpdt                         (NcoI/EcoRI, 992+10+6, L, pcrdig)

Digest pBAD-myc-his-A                 (NcoI/EcoRI, 4053+41, L, vectdig)

Ligate pcrdig and vectdig             (pBAD-TrpRS)

--

>TrpRS-Fwd	Forward primer for PCR of TrpRS from Thermotoga Maritima  

ccagtCCATGGcaAGAATACTGAGCGGCATGAG

>TrpRS-Rv	Reverse primer for PCR of TrpRS from Thermotoga Maritima 

ccagtGAATTCTCAGAACATCAGGTTCATG

>TrpRS gene DNA sequence in Thermotoga Maritima genome

TTGAGAATACTGAGCGGCATGAGACCTACCGGAAAACTCCATATAGGTCATCTCGTGGGAGCTCTGGAAA
ACTGGGTGAAGCTTCAGGAAGAAGGAAACGAATGTTTCTACTTTGTCGCGGATTGGCACGCTTTGACCAC
CCACTACGACGATGTTTCGAAGCTCAAAGAATACACCCGCGACCTGGTGAGGGGATTTCTCGCCTGTGGA
ATAGATCCTGAAAAGTCCGTGATTTTTGTTCAGTCTGGTGTCAAAGAGCACGCTGAGCTTGCACTGCTTT
TCAGCATGATCGTTTCTGTTTCACGTCTCGAGAGGGTTCCCACTTACAAAGAGATAAAAAGTGAGCTGAA
CTACAAAGATCTTTCCACGGCTGGTTTTCTCATCTATCCCGTTCTTCAGGCAGCCGATATTTTGATCTAC
AAAGCTGAAGGAGTACCAGTCGGTGAAGATCAGGTTTACCACATAGAACTCACGAGGGAGATCGCCAGGC
GTTTTAACTATCTCTACGATGAAGTCTTTCCAGAACCAGAAGCAATTCTGTCTCGGGTTCCAAAGCTTCC
AGGAACGGACGGCCGAAAGATGAGCAAAAGCTATGGGAACATAATAAACCTAGAAATCTCGGAAAAAGAA
CTGGAACAGACGATACTGAGGATGATGACCGATCCAGCGAGGGTGAGAAGGAGCGACCCTGGAAATCCGG
AGAACTGCCCCGTATGGAAATACCACCAGGCGTTCGACATCAGTGAAGAAGAGAGCAAATGGGTATGGGA
AGGCTGTACAACGGCCAGCATCGGCTGTGTTGATTGTAAGAAGTTGCTGTTGAAGAATATGAAACGAAAA
TTGGCACCGATCTGGGAGAACTTCAGAAAAATAGACGAAGATCCACACTACGTGGACGACGTTATAATGG
AAGGGACGAAGAAGGCCAGAGAAGTGGCTGCTAAAACGATGGAAGAAGTGAGAAGGGCCATGAACCTGAT
GTTCTGA

>pBAD-myc-his-A  
AAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTGGGCTAACAGGAGGAATTAACCATGGATCCGAGCTCGAGATCTGCAGCTGGTACCATATGGGAATTCGAAGCTTGGGCCCGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCAGATCAATTCGCGCGCGAAGGCGAAGCGGCATGCATAATGTGCCTGTCAAATGGACGAAGCAGGGATTCTGCAAACCCTATGCTACTCCGTCAAGCCGTCAATTGTCTGATTCGTTACCAATTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGATATCAAAATTGCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGAACCCCGTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGCCAGTAGGCGCGCGGACGAAAGTAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGATGAATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTTCACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGAGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAG

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Construction file for TyrRS

PCR TyrRS-Fwd/TyrRS-Rv on Thermotoga Maritima  (1248 bp, pcrpdt)

PCR TyrRS-Fwd/NcoI-SOEing-Rv on pcrpdt  (922 bp, A)

PCR NcoI-SOEing-Fwd/TyrRS-Rv on pcrpdt  (352bp, B)

PCR TyrRS-Fwd/TyrRS-Rv on A+B		(1248 bp, newpcrpdt)

Digest newpcrpdt                        (NcoI/EcoRI, 1232+10+6, L, pcrdig)

Digest pBAD-myc-his-A                   (NcoI/EcoRI, 4053+41, L, vectdig)

Ligate pcrdig and vectdig               (pBAD-TyrRS)

--

>TyrRS-Fwd	Forward primer for PCR of TyrRS from Thermotoga Maritima  

ccagtCCATGGcaacgccggaggaacaggtg

>TyrRS-Rv	Reverse primer for PCR of TyrRS from Thermotoga Maritima 

ccagtGAATTCcaaaggaaattcaagagttttc

>NcoI-SOEing-Fwd	Forward primer for SOEing PCR of TyrRS from Thermotoga Maritima 
 
cgcgcttcttCCAcGGtgaagaaaac

>NcoI-SOEing-Rv 	Reverse primer for SOEing PCR of TyrRS from Thermotoga Maritima 

gttttcttcaCCgTGGaagaagcgcg

>TyrRS gene DNA sequence in Thermotoga Maritima genome 

ATGACGCCGGAGGAACAGGTGAAAATTCTCAAAAGAAACGTTGTTGACCTCATAAGTGAAGAAGAACTCCTCGACAGAATAAAAAGAAAAGGAAAACTCCGCGTGAAACTCGGTGTGGATCCCTCAAGGCCCGATTTGCATCTGGGTCACGCGGTCGTTCTGAGGAAGTTGAGAGAATTTCAGGATCTCGGTCACACGGTCGTTCTGATCATAGGAGACTTCACCGCACGTATTGGTGATCCCTCCGGAAGAAACGAAACACGCCCCATGCTGACCAAAGAAGAGGTTCTGGAGAACGCAAAGACCTATCAGGAGCAGGCCTTCAAAATACTGGATCCCAAAAGAACGGAACTTCGCTTCAACGGTGAGTGGCTCGACAGGATGACCTTCGCAGATGTGATCATTCTGGCTTCGAAGTACACGGTTGCGAGGATGCTCGAGAGAGACGATTTCGCAAAGAGATTCAAAGAAGGCATTCCCATTGCCATATCAGAGTTTCTGTATCCGCTCGCACAGGCCTACGATTCCGTTGCCATCCAGTCAGATGTGGAACTCGGCGGAACGGATCAGCTTTTCAACCTCCTTGTGGGAAGGAAGATACAGGAAGAATACGGTCAAGAGCCCCAGATCGTCATGACGATGCCGATCATCGAGGGAACAGACGGAAAATTGAAGATGAGCAAAAGCTACGGAAACTACATCGCTTTCAACGATCCGCCCGAGGAGATGTACGGCAAACTCATGTCCATACCTGATGAACTCATCATAAAATACATGCGCCTTCTCACGGACATCCCAGAAGAACGGATCGAAGAGTACGAAAGAAAGATGAAGGAAAAAACGATCAATCCACGAGACGTGAAGATGGTTCTCGCGTACGAGATAACGCGCTTCTTCCATGGTGAAGAAAACGCAAAGAAGGCCCAGGAACACTTCGTGAAAGTCTTTCAAAAGAAAGAAATTCCTGACGAGATGCCGGTCGTTGAGATTTCTCAGGAGAAGAACATCGTGGATCTCCTCGTGGAGATAGGAGCTGCATCCAGCAAAAGTGAGGCTAAAAGACTCGTTTCTCAAGGTGGAGTGTACATCGACGGAGAGAGGATAGAGGACATAAAATTCACTGTAGAACCTGATGGAGAGCGAGTTTTGAGAGTTGGAAAGAGGAAGTTCTACAGAATATCAGGTGGAGAGACAAAAAAACTTTAGAAAACTCTTGAATTTCCTTTG

>pBAD-myc-his-A  
AAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTGGGCTAACAGGAGGAATTAACCATGGATCCGAGCTCGAGATCTGCAGCTGGTACCATATGGGAATTCGAAGCTTGGGCCCGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCAGATCAATTCGCGCGCGAAGGCGAAGCGGCATGCATAATGTGCCTGTCAAATGGACGAAGCAGGGATTCTGCAAACCCTATGCTACTCCGTCAAGCCGTCAATTGTCTGATTCGTTACCAATTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGATATCAAAATTGCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGAACCCCGTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGCCAGTAGGCGCGCGGACGAAAGTAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGATGAATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTTCACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGAGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAG

February 23rd

Karim R. Merchant 01:47, 24 February 2014 (EST)

Today I spent the day checking my group's primers and primers and making necessary changes, along with making a compilation file with all the primers our group needs listed. A list of changes I made can be found in the following text, which is a copy of the email I sent to my group.

"Hey guys,

I have attached a complete primer list. Please check.

Chris- I redid your ArgRS and SerRS primers to have BsaI on both ends. This way you don't have to do a double digest, and also don't have to do SOEing, as there are no BsaI sites in ArgRS (or SerRS).

Tae Won- I did your GlnRS SOEing primers so they don't change your amino acid sequence.

Kelvin- For SOEing, the internal forward and reverse primers have to be compliments to each other. I redid your ThrRS internal SOEing primers. Also, I changed the "extra sequence" that comes in front of the restriction site for Valyl External Forward to no longer have an ATG in it. It shouldn't matter of course, as it's supposed to be cut out. Just a precaution.

Christy- your second oligo was the reverse compliment of what the primer should be so I switched it for you.

Everyone needs to check that their primers work and don't mess up the amino acid sequence for their respective protein, other than perhaps adding the extra Alanine after the initial Methionine. Also, please update your construction files with these primers/processes. Some proteins have shifted from SOEing to using BsaI. Please reply back to this email ASAP to confirm primers work, so I can send this file to Professor Anderson."

The compilation file I made with all the primers listed is attached: Media:Group4Primers.docx

February 25th

Karim R. Merchant 00:38, 26 February 2014 (EST)

Today we checked and finalized our primers during lab. They are attached: Media:Group_4_Primers.xlsx

I also updated my construction file for TyrRS without SOEing, and with BsaI sites:

Construction file for TyrRS

PCR TyrRS BsaI NcoI Fwd/TyrRS BsaI EcoRI Rv on Thermotoga Maritima  (1260 bp, pcrpdt)
Digest pcrpdt                        	(BsaI, 1232+16+12, L, pcrdig)
Digest pBAD-myc-his-A                   (NcoI/EcoRI, 4053+41, L, vectdig)
Ligate pcrdig and vectdig               (pBAD-TyrRS)
-----------------------------------------
>TyrRS BsaI NcoI Fwd	Forward primer for PCR of TyrRS from Thermotoga Maritima  
ccagtGGTCTCCCATGGcaacgccggaggaacaggtg
>TyrRS BsaI EcoRI Rv	Reverse primer for PCR of TyrRS from Thermotoga Maritima 
ccagtGGTCTCGAATTCcaaaggaaattcaagagttttc
>TyrRS gene DNA sequence in Thermotoga Maritima genome
ATGACGCCGGAGGAACAGGTGAAAATTCTCAAAAGAAACGTTGTTGACCTCATAAGTGAAGAAGAACTCCTCGACAGAATAAAAAGAAAAGGAAAACTCCGCGTG
AAACTCGGTGTGGATCCCTCAAGGCCCGATTTGCATCTGGGTCACGCGGTCGTTCTGAGGAAGTTGAGAGAATTTCAGGATCTCGGTCACACGGTCGTTCTGATC
ATAGGAGACTTCACCGCACGTATTGGTGATCCCTCCGGAAGAAACGAAACACGCCCCATGCTGACCAAAGAAGAGGTTCTGGAGAACGCAAAGACCTATCAGGAG
CAGGCCTTCAAAATACTGGATCCCAAAAGAACGGAACTTCGCTTCAACGGTGAGTGGCTCGACAGGATGACCTTCGCAGATGTGATCATTCTGGCTTCGAAGTAC
ACGGTTGCGAGGATGCTCGAGAGAGACGATTTCGCAAAGAGATTCAAAGAAGGCATTCCCATTGCCATATCAGAGTTTCTGTATCCGCTCGCACAGGCCTACGAT
TCCGTTGCCATCCAGTCAGATGTGGAACTCGGCGGAACGGATCAGCTTTTCAACCTCCTTGTGGGAAGGAAGATACAGGAAGAATACGGTCAAGAGCCCCAGATC
GTCATGACGATGCCGATCATCGAGGGAACAGACGGAAAATTGAAGATGAGCAAAAGCTACGGAAACTACATCGCTTTCAACGATCCGCCCGAGGAGATGTACGGC
AAACTCATGTCCATACCTGATGAACTCATCATAAAATACATGCGCCTTCTCACGGACATCCCAGAAGAACGGATCGAAGAGTACGAAAGAAAGATGAAGGAAAAA
ACGATCAATCCACGAGACGTGAAGATGGTTCTCGCGTACGAGATAACGCGCTTCTTCCATGGTGAAGAAAACGCAAAGAAGGCCCAGGAACACTTCGTGAAAGTC
TTTCAAAAGAAAGAAATTCCTGACGAGATGCCGGTCGTTGAGATTTCTCAGGAGAAGAACATCGTGGATCTCCTCGTGGAGATAGGAGCTGCATCCAGCAAAAGT
GAGGCTAAAAGACTCGTTTCTCAAGGTGGAGTGTACATCGACGGAGAGAGGATAGAGGACATAAAATTCACTGTAGAACCTGATGGAGAGCGAGTTTTGAGAGTT
GGAAAGAGGAAGTTCTACAGAATATCAGGTGGAGAGACAAAAAAACTTTAGAAAACTCTTGAATTTCCTTTG

>pBAD-myc-his-A  
AAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTGGGCTAACAGGAGGAATTAACCATGGATCCGAGCTCGAGATCTGCAGCTGGTACCATATGGGAATTCGAAGCTTGGGCCCGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCAGATCAATTCGCGCGCGAAGGCGAAGCGGCATGCATAATGTGCCTGTCAAATGGACGAAGCAGGGATTCTGCAAACCCTATGCTACTCCGTCAAGCCGTCAATTGTCTGATTCGTTACCAATTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGATATCAAAATTGCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGAACCCCGTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGCCAGTAGGCGCGCGGACGAAAGTAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGATGAATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTTCACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGAGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAG

February 27th

Karim R. Merchant 15:34, 28 February 2014 (EST)

Today we all did minipreps to collect plasmid pBAD-myc-his-A from e. coli cells. I did 2x 1.5 mL cell juice minipreps. The protocol can be found here: JCA Protocols

March 4th

Karim R. Merchant 02:14, 5 March 2014 (EST)

Today I ran my first PCRs to amplify the genes TrpRS and TyrRS out of the Thermotoga Maritima genome. First I diluted the stock primers to 100 uM and then made a dilution of that to 10 uM. For TrpRS, my primers were TrpRS NcoI Fwd/TrpRS EcoRI Rv, and for TyrRS my primers were TyrRS BsaI NcoI Fwd/TyrRS BsaI EcoRI Rv. Both PCRs were below 2 kilobases in length.

For both PCRs, I followed the following mixture setup:

24 uL ddH2O

3.3 uL Expand Buffer 2

3.3 uL dNTP 2mM in each stock

1 uL 10 uM primer 1

1 uL 10 uM primer 2

0.5 uL genomic DNA

0.5 uL Expand Polymerase 1

=33.6 uL total

The Thermocycler set up was:

1. 94°C for 2 min

2. 94°C for 15 sec

3. 55°C for 30 sec (should be lower than Tm of all primers)

4. 72°C for 2 min (1 min/kilobase)

5. Go to step 2 30x

6. 72°C for 7 min

7. 16°C for ever.

Next: Gel clean ups are on Thursday March 6th

March 6th

Karim R. Merchant 20:22, 9 March 2014 (EDT)

Today we ran an analytical agarose gel to see the sizes of our group's PCR products. The agarose gel was made by mixing 1g of agarose powder in 1mL of 1x TAE buffer, and microwaved until it reached a boiling state. Next, 10 uL of 10,000x Gel dye was added. The samples were prepared by mixing 2 uL of the PCR product with 6 uL of ddH2O along with 1 uL of DNA loading dye. The first lane of the gel was loaded with 5 uL of Quick Load 2-Log DNA ladder.

The gel picture is below:

1st lane = DNA ladder

2nd lane = TrpRS product, should be 1008 bp

3rd lane = TyrRS product, should be 1260 bp

4th lane = ArgRS product, Chris's product

5th lane = SerRS product, Chris's product

6th lane = GlnRS subunit C product, Tae's product

March 11th

Karim R. Merchant 22:26, 13 March 2014 (EDT)

Today I redid my TrpRS PCR using the product of the TrpRS PCR I did week as template. I did this due to the fact that my product concentration seemed a little low based on the band intensity on the March 6th gel. I used the exact same reaction mixture and thermocycle set-up as is written on the March 4th notebook entry. The only difference was that the PCR template DNA was not the Thermotoga Maritima genome, but the original TrpRS PCR product, which had undergone Zymogen DNA cleanup.

March 13th

Karim R. Merchant 22:26, 13 March 2014 (EDT)

Today I ran a gel of my second TrpRS PCR, and also did the Zymogen DNA cleanup for it. The image is below:

Media:13032014 BioE 140L Group 4 Gel.jpg

First lane is the Quick Load 2-Log DNA ladder. In the second lane was my product: TrpRS should be 1008 bp, so it looks like it's in the right spot. It's also quite bright, indicating there's a lot of the product. The only bad side was that the gel had a "smile", and the lanes all curved. At least the gel was happy.

March 18th

Karim R. Merchant 23:31, 18 March 2014 (EDT)

Today I did 3 digestions, and a gel clean-up of the digested products. The reactions were as below:

TrpRS Digestion:

8 uL TrpRS2 PCR product

1 uL NEB Buffer 2

0.5 uL EcoRI

0.5 uL NcoI

=10 uL

TyrRS Digestion:

8 uL TyrRS PCR product

1 uL NEB CutSmart Buffer

0.5 uL BsaI

0.5 uL ddH2O

=10 uL

pBAD myc-his-A plasmid vector Digestion:

15 uL vector mini-prep product

3 uL NEB Buffer 2

1.5 uL EcoRI

1.5 uL NcoI

9 uL ddH2O

=30 uL

I centrifuged the reaction mixtures and left them at 37 degrees Celsius for 1 hour.

Next I did a gel purification clean up with ZymoGen. The protocol can be found here: JCA Protocols

An image of the gel is here:

The first lane is Quick load 2 log DNA ladder. The second lane is TrpRS digestion product. The third lane is TyrRS digestion product. The next 3 lanes are all vector digestion products. TrpRS should have a band at 992 bp after digestion, as it does. TyrRS should have a band at 1232 bp after digestion, as it does. The vector digest should have a band at 4053 bp, as it does. All digests went smoothly. Next step is ligation and transformation. TrpRS and TyrRS digestions were eluted after gel cleanup in 10 uL of water, and the the vector digest was eluted in 15 uL.

April 1st

Karim R. Merchant 00:42, 4 April 2014 (EDT)

Today I did the ligation of TrpRS and TyrRS with the pBAD myc his A vector backbone.

There were 3 ligations:

TrpRS and TyrRS:

6.5 uL ddH2O

1 uL T4 DNA Ligase Buffer

1 uL pBAD digested backbone

1 uL digested insert

0.5 uL T4 DNA Ligase

--

10uL

Negative control:

7.5 uL ddH2O

1 uL T4 DNA Ligase Buffer

1 uL pBAD digested backbone

0.5 uL T4 DNA Ligase

--

10uL

I mixed and centrifuged all the ligations and left them at room temperature for 30 minutes.

Next I transformed all 3 of them.

I added 35 uL of KCM to 3 alliquots of 100 uL cells. Next I cooled them for 1 minute. Then I added 65 uL of the KCM+cell mixture to each 10 uL ligation product. I cooled all the cells on ice for 10 minutes. Then I heat shocked the cells at 42 degrees Celsius for 90 seconds (the heat block said 42 but the thermometer read 37). Next I cooled the cells for 1 minute. Then I did a rescue step where i added 100 uL of LB media to each cell alliquot and shook it at 37 degrees celsius for 1 hour. Finally I plated 100 uL of cell juice on ampicillin plates. Total = 3 plates = TrpRS, TyrRS, and negative control.

April 3rd

Karim R. Merchant 00:49, 4 April 2014 (EDT)

None of the plates from last time had any colonies. The professor and I think the heat shock failed. We agreed on 3 modifications for next time: use double the amount of insert during the ligations, use water in the heat shock block or better yet use the thermocycler, and finally also do a positive control using the miniprep plasmid with 0.5 uL of plasmid DNA in the transformation.

I was unable to do this today as there were not enough cells in lab. I will do this on Tuesday.