Team:British Columbia/New Biobricks

From 2012.igem.org

Latest revision as of 05:08, 28 June 2012

Site-directed Mutagenesis

Jacob, Grace, and Ting-Chia will be working on getting rid of illegal sites within biobricks.

Marianne and Ruichen will be working on this as well (Monday Night)

I suggest that we just synthesize the dszA (4 PstI sites). Saves us time and grief. -Jacob

While teaching Ruichen and Mehul how to design mutagenesis primers, I came up with the primers for getting rid of the PstI from dszB. Here they are:

Primer pair 1. Getting rid of the PstI from dszB.

   Forward: 5' CAACAACTTGCTACAGGAACCCGTC 3'
   Reverse: 5' GACGGGTTCCTGTAGCAAGTTGTTG 3'
                           
    GC content: 52.00%           Location: 24-48
    Melting temp: 71.8°C         Mismatched bases: 1
    Length: 25 bp                Mutation: Substitution
    5' flanking region: 12 bp    Forward primer MW: 7580.05 Da
    3' flanking region: 12 bp    Reverse primer MW: 7744.11 Da

Primer paire 2. Getting rid of the NotI from dszB.

   Forward: 5' GACAGCCCGATCACAACGGCCGCCGACCTTG 3'
   Reverse: 5' CAAGGTCGGCGGCCGTTGTGATCGGGCTGTC 3'
                              
    GC content: 67.74%           Location: 52-82
    Melting temp: 84.6°C         Mismatched bases: 1
    Length: 31 bp                Mutation: Substitution
    5' flanking region: 15 bp    Forward primer MW: 9436.24 Da
    3' flanking region: 15 bp    Reverse primer MW: 9600.30 Da

- Ruichen

Primer pair 3. Getting rid of the PstI at (487) from dszC

   Forward: 5' CGAAGCACTTCTACAGCGGCGCCAAG 3'
   Reverse: 5' CTTGGCGCCGCTGTAGAAGTGCTTCG 3'
                            
    GC content: 61.54%           Location: 23-48
    Melting temp: 77.0°C         Mismatched bases: 1
    Length: 26 bp                Mutation: Substitution
    5' flanking region: 12 bp    Forward primer MW: 7950.27 Da
    3' flanking region: 13 bp    Reverse primer MW: 7994.27 Da

Primer Pair 4. Getting rid of the PstI at (971) from dszC

   Forward: 5' GGCCCACCTGCTACAGACGGTGTGG 3'
   Reverse: 5' CCACACCGTCTGTAGCAGGTGGGCC 3'
                           
    GC content: 68.00%           Location: 42-66
    Melting temp: 78.4°C         Mismatched bases: 1
    Length: 25 bp                Mutation: Substitution
    5' flanking region: 12 bp    Forward primer MW: 7684.07 Da
    3' flanking region: 12 bp    Reverse primer MW: 7644.05 Da

- Ruichen

Amino acid gene sequences: TyrA:

ATGGTTGCTGAATTGACCGCATTACGCGATCAAATTGATGAAGTCGATAAAGCGCTGCTGAATTTATTAGCGAAGCGTCTGGAACTGGTTGCTGAAGTGGGCGAGGTGAAAAGCCGCTTTGGACTGCCTATTTATGTTCCGGAGCGCGAGGCATCTATGTTGGCCTCGCGTCGTGCAGAGGCGGAAGCTCTGGGTGTACCGCCAGATCTGATTGAGGATGTTTTGCGTCGGGTGATGCGTGAATCTTACTCCAGTGAAAACGACAAAGGATTTAAAACACTTTGTCCGTCACTGCGTCCGGTGGTTATCGTCGGCGGTGGCGGTCAGATGGGACGCCTGTTCGAGAAGATGCTGACCCTCTCGGGTTATCAGGTGCGGATTCTGGAGCAACATGACTGGGATCGAGCGGCTGATATTGTTGCCGATGCCGGAATGGTGATTGTTAGTGTGCCAATCCACGTTACTGAGCAAGTTATTGGCAAATTACCGCCTTTACCGAAAGATTGTATTCTGGTCGATCTGGCATCAGTGAAAAATGGGCCATTACAGGCCATGCTGGTGGCGCATGATGGTCCGGTGCTGGGGCTACACCCGATGTTCGGTCCGGACAGCGGTAGCCTGGCAAAGCAAGTTGTGGTCTGGTGTGATGGACGTAAACCGGAAGCATACCAATGGTTTCTGGAGCAAATTCAGGTCTGGGGCGCTCGGCTGCATCGTATTAGCGCCGTCGAGCACGATCAGAATATGGCGTTTATTCAGGCACTGCGCCACTTTGCTACTTTTGCTTACGGGCTGCACCTGGCAGAAGAAAATGTTCAGCTTGAGCAACTTCTGGCGCTCTCTTCGCCGATTTACCGCCTTGAGCTGGCGATGGTCGGGCGACTGTTTGCTCAGGATCCGCAGCTTTATGCCGACATCATTATGTCGTCAGAGCGTAATCTGGCGTTAATCAAACGTTACTATAAGCGTTTCGGCGAGGCGATTGAGTTGCTGGAGCAGGGCGATAAGCAGGCGTTTATTGACAGTTTCCGCAAGGTGGAGCACTGGTTCGGCGATTACGCACAGCGTTTTCAGAGTGAAAGCCGCGTGTTATTGCGTCAGGCGAATGACAATCGCCAGTAA

MetA:

atgCCGATTCGTGTGCCGGACGAGCTACCCGCCGTCAATTTCTTGCGTGAAGAAAACGTCTTTGTGATGACAACTTCTCGTGCGTCTGGTCAGGAAATTCGTCCACTTAAGGTTCTGATCCTTAACCTGATGCCGAAGAAGATTGAAACTGAAAATCAGTTTCTGCGCCTGCTTTCAAACTCACCTTTGCAGGTCGATATTCAGCTGTTGCGCATCGATTCCCGTGAATCGCGCAACACG CCCGCAGAGCATCTGAACAACTTCTACTGTAACTTTGAAGATATTCAGGATCAGAACTTTGACGGTTTGATTGTAACTGGTGCGCCGCTGGGCCTGGTGGAGTTTAATGATGTCGCTTAC TGGCCGCAGATCAAACAGGTGCTGGAGTGGTCGAAAGATCACGTCACCTCGACGCTGTTTGTCTGCTGGGCGGTACAGGCCGCGCTCAATATCCTCTACGGCATTCCTAAGCAAACTCGCACCGAAAAACTCTCTGGCGTTTACGAGCATCATATTCTCCATCCTCATGCGCTTCTGACGCGTGGCTTTGATGATTCATTCCTGGCACCGCATTCGCGCTATGCTGACTTTCCGGCAGCG TTGATTCGTGATTACACCGATCTGGAAATTCTGGCAGAGACGGAAGAAGGGGATGCATATCTGTTTGCCAGTAAAGATAAGCGCATTGCCTTTGTGACGGGCCATCCCGAATATGATGCGCAAACGCTGGCGCAGGAATTTTTCCGCGATGTGGAAGCCGGACTAGACCCGGATGTACCGTATAACTATTTCCCGCACAATGATCCGCAAAATACACCGCGAGCGAGCTGGCGTAGTCACGGTAATTTACTGTTTACCAACTGGCTCAACTATTACGTCTACCAGATCACGCCATACGATCTACGGCACATGAATCCAACGCTGGATtaa

TrpA:

atgGAACGCT ACGAATCTCT GTTTGCCCAG TTGAAGGAGC GCAAAGAAGG CGCATTCGTT CCTTTCGTCA CGCTCGGTGA TCCGGGCATT GAGCAGTCAT TGAAAATTAT CGATACGCTA ATTGAAGCCG GTGCTGACGC GCTGGAGTTA GGTATCCCCT TCTCCGACCC ACTGGCGGAT GGCCCGACGA TTCAAAACGC CACTCTGCGC GCCTTTGCGG CAGGTGTGAC TCCGGCACAA TGTTTTGAAA TGCTGGCACT GATTCGCCAG AAACACCCGA CCATTCCCAT TGGCCTGTTG ATGTATGCCA ATCTGGTGTT TAACAAAGGC ATTGATGAGT TTTATGCCCA GTGCGAAAAA GTCGGCGTCG ATTCGGTGCT GGTTGCCGAT GTGCCAGTTG AAGAGTCCGC GCCCTTCCGC CAGGCCGCGT TGCGTCATAA TGTCGCACCT ATCTTCATCT GCCCGCCAAA TGCCGATGAC GACCTGCTGC GCCAGATAGC CTCTTACGGT CGTGGTTACA CCTATTTGCT GTCACGAGCA GGCGTGACCG GCGCAGAAAA CCGCGCCGCG TTACCCCTCA ATCATCTGGT TGCGAAGCTG AAAGAGTACA ACGCTGCACC TCCATTGCAG GGATTTGGTA TTTCCGCCCC GGATCAGGTA AAAGCAGCGA TTGATGCAGG AGCTGCGGGC GCGATTTCTG GTTCGGCCAT TGTTAAAATC ATCGAGCAAC ATATTAATGA GCCAGAGAAA ATGCTGGCGG CACTGAAAGT TTTTGTACAA CCGATGAAAG CGGCGACGCG CAGTtaa

TrpB:

    atgACAACAT TACTTAACCC CTATTTTGGT GAGTTTGGCG GCATGTACGT GCCACAAATC

CTGATGCCTG CTCTGCGCCA GCTGGAAGAA GCTTTTGTCA GTGCGCAAAA AGATCCTGAA TTTCAGGCTC AGTTCAACGA CCTGCTGAAA AACTATGCCG GGCGTCCAAC CGCGCTGACC AAATGCCAGA ACATTACAGC CGGGACGAAC ACCACGCTGT ATCTCAAGCG TGAAGATTTG CTGCACGGCG GCGCGCATAA AACTAACCAG GTGCTGGGGC AGGCGTTGCT GGCGAAGCGG ATGGGTAAAA CCGAAATCAT CGCCGAAACC GGTGCCGGTC AGCATGGCGT GGCGTCGGCC CTTGCCAGCG CCCTGCTCGG CCTGAAATGC CGTATTTATA TGGGTGCCAA AGACGTTGAA CGCCAGTCGC CTAACGTTTT TCGTATGCGC TTAATGGGTG CGGAAGTGAT CCCGGTGCAT AGCGGTTCCG CGACGCTGAA AGATGCCTGT AACGAGGCGC TGCGCGACTG GTCCGGTAGT TACGAAACCG CGCACTATAT GCTGGGCACC GCAGCTGGCC CGCATCCTTA TCCGACCATT GTGCGTGAGT TTCAGCGGAT GATTGGCGAA GAAACCAAAG CGCAGATTCT GGAAAGAGAA GGTCGCCTGC CGGATGCCGT TATCGCCTGT GTTGGCGGCG GTTCGAATGC CATCGGCATG TTTGCTGATT TCATCAATGA AACCAACGTC GGCCTGATTG GTGTGGAGCC AGGTGGTCAC GGTATCGAAA CTGGCGAGCA CGGCGCACCG CTAAAACATG GTCGCGTGGG TATCTATTTC GGTATGAAAG CGCCGATGAT GCAAACCGAA GACGGGCAGA TTGAAGAATC TTACTCCATC TCCGCCGGAC TGGATTTCCC GTCTGTCGGC CCACAACACG CGTATCTTAA CAGCACTGGA CGCGCTGATT ACGTGTCTAT TACCGATGAT GAAGCCCTTG AAGCCTTCAA AACGCTGTGC CTGCACGAAG GGATCATCCC GGCGCTGGAA TCCTCCCACG CCCTGGCCCA TGCGTTGAAA ATGATGCGCG AAAACCCGGA TAAAGAGCAG CTACTGGTGG TTAACCTTTC CGGTCGCGGC GATAAAGACA TCTTCACCGT TCACGATATT TTGAAAGCAC GAGGGGAAAT Ctga

ArgC:

     atgTTGAATA CGCTGATTGT GGGTGCCAGC GGCTACGCTG GCGCAGAGCT AGTGACCTAT

GTAAATCGCC ATCCGCATAT GAACATAACC GCTTTGACTG TTTCAGCGCA AAGCAATGAT GCGGGAAAGT TAATCTCCGA TTTGCATCCG CAGCTAAAAG GCATCGTTGA TCTGCCGTTG CAGCCGATGT CGGATATCAG CGAGTTTAGC CCAGGGGTGG ACGTAGTGTT TCTCGCCACC GCCCATGAAG TTAGCCACGA TTTAGCGCCG CAGTTTCTTG AAGCGGGCTG CGTGGTGTTC GACCTTTCCG GCGCGTTTCG TGTTAACGAC GCCACCTTCT ATGAAAAATA TTACGGCTTT ACCCATCAAT ACCCGGAACT GTTGGAACAG GCAGCCTACG GTCTGGCGGA GTGGTGCGGT AATAAATTAA AAGAAGCGAA TTTGATTGCG GTGCCGGGCT GTTATCCGAC GGCGGCACAG CTGGCGCTGA AACCGTTGAT TGATGCCGAT CTTCTTGACC TCAATCAGTG GCCGGTGATC AACGCCACCA GCGGCGTGAG CGGTGCAGGG CGTAAAGCGG CCATTTCAAA CAGCTTTTGT GAAGTTAGCC TGCAACCGTA TGGCGTCTTT ACTCATCGCC ATCAACCAGA GATCGCCACA CACCTCGGTG CTGACGTTAT CTTCACCCCA CATCTGGGCA ATTTCCCGCG CGGCATTCTC GAAACCATTA CCTGCCGCCT GAAATCGGGT GTGACCCAGG CGCAAGTCGC GCAAGTGTTA CAGCAGGCGT ATGCCCATAA ACCGCTGGTG CGGCTGTATG ACAAAGGCGT TCCGGCGCTG AAAAATGTCG TTGGGCTGCC ATTTTGCGAT ATCGGGTTTG CCGTTCAGGG CGAGCATCTG ATTATTGTGG CGACCGAAGA CAACTTACTG AAAGGCGCGG CGGCACAAGC GGTACAGTGC GCCAATATTC GTTTCGGCTA TGCGGAAACG CAGTCTCTTA TTtaa

ArgE:

     atgAAAAACA AATTACCGCC ATTTATCGAG ATTTACCGCG CTCTGATTGC CACACCTTCA

ATAAGCGCCA CGGAAGAGGC ACTCGATCAA AGCAATGCAG ATTTAATCAC TCTGCTGGCG GACTGGTTTA AAGATTTGGG CTTCAATGTG GAAGTGCAGC CTGTTCCAGG AACTCGCAAC AAATTCAATA TGCTGGCAAG TATCGGACAG GGGGCTGGCG GCTTGTTGCT GGCGGGGCAT ACCGATACGG TGCCATTTGA TGACGGTCGC TGGACGCGCG ATCCGTTTAC ACTGACGGAG CATGACGGCA AGCTTTACGG CTTAGGCACC GCCGACATGA AAGGCTTTTT TGCGTTTATC CTTGATGCGC TACGCGATGT CGACGTCACG AAACTGAAAA AACCGCTCTA CATTCTGGCG ACTGCTGATG AAGAAACCAG TATGGCCGGA GCGCGTTATT TTGCCGAAAC TACCGCCCTG CGCCCGGATT GCGCCATCAT TGGCGAACCG ACGTCACTAC AACCGGTACG CGCACATAAA GGTCATATCT CTAACGCCAT CCGTATTCAG GGCCAGTCGG GGCACTCCAG CGATCCAGCA CGCGGAGTTA ACGCTATCGA ACTAATGCAC GACGCCATCG GGCATATTTT GCAATTGCGC GATAACCTGA AAGAACGTTA TCACTACGAA GCGTTTACCG TGCCATACCC TACGCTCAAC CTCGGGCATA TTCACGGTGG CGACGCTTCT AACCGTATTT GCGCTTGCTG TGAGTTGCAT ATGGATATTC GTCCGCTGCC TGGCATGACA CTCAATGAAC TTAATGGTTT GCTCAACGAT GCATTGGCTC CGGTGAGCGA ACGCTGGCCG GGTCGTCTGA CGGTCGACGA GCTGCATCCG CCGATCCCTG GCTATGAATG CCCACCGAAT CATCAACTGG TTGAAGTGGT TGAGAAATTG CTCGGAGCAA AAACCGAAGT GGTGAACTAC TGTACCGAAG CGCCGTTTAT TCAAACGTTA TGCCCGACGC TGGTGTTGGG GCCTGGCTCA ATTAATCAGG CTCATCAACC TGATGAATAT CTGGAAACAC GGTTTATCAA GCCCACCCGC GAACTGATAA CCCAGGTAAT TCACCATTTT TGCTGGCATt aa

June 21

Jacob and I are working on getting amino acid genes into biobricks. We're on our fourth (?) ligation with some of them because they haven't been working... We suspect the ligation step has been causing the trouble and we altered the protocol slightly - 3:1 insert to vector ratio (in uL) as opposed to the 2:2 used previously, and using NEB Buffer 3 as opposed to Buffer 2 (to optimize EcoR1 and PST1 activity). We also left the ligation mix sitting at room temperature beginning at 5 pm overnight. Jacob will transform the cloned vectors into competent cells (K12?) and plate them tomorrow morning (~9 am). This increases the ligation process from 1.5 hours (June 20) to 16 hours.

The genes I digested and ligated:

• Trp A
• Trp B
• Tyr A
• Met A
• Arg C

Jacob and I digested/ligated/transformed those listed above yesterday as well, but Arg C has replaced Arg E today because the Arg E cells grew several (~5) colonies! Success!

I also (re)ligated the pSB1C3 vector left over from last year (about 8 months old). It has the RFP gene cloned into it. This was to (1) amplify the number of cloned vectors, and (2) to check if our ligation protocol is effective.

Joe was digesting 2 of the dsz genes and planned to ligate tomorrow.

- Grace.yi

June 22

Jacob found one colony on the MetA plate from the cloning done previously (before the 21st). To do: inoculate a culture, miniprep, sequence! Likewise for the ArgE.

The cloned vectors from yesterday were transformed into DH5a and plated on Chlor plates.

June 23

Appears to be no colonies on any of the plates made yesterday. Will keep them in the 37° room in case colonies appear later.

June 25

No colonies on the plates made on June 22 - need to redo. Inoculated 3 mL cultures of LB with colonies of the ArgE biobrick and the pSB1C3 vector cloned with RFP. This will be miniprepped tomorrow.

A restriction digest was set up to test the efficiency of our EcoR1-HF (we do not have plain EcoR1) and Pst1 enzymes, because cloning has been unsuccessful thus far. Two digest mixes were made: (1) the EcoR1 and Pst1 with later expiry dates, and (2) the EcoR1 and Pst1 with earlier or passed expiry dates. The DNA template used was a biobrick, 3I. After being run through the thermocycler, the completed digests were stored in the -20°C freezer. They will be run through a gel by Joe tomorrow, and the size of the products will indicate whether or not the digest was successful.

The protocols we have been using for Restriction Digests and Ligations have been put up on the wiki.

- grace.yi

June 27

Miniprepping of the ArgE and pSB1C3+RFP was done today. Elution was done with water and not elution buffer so the products could be sent for sequencing. However, the incorrect amount of water was added (75 uL) and the plasmid concentration well below the needed concentration of ~200 ng/uL - need to evaporate water to concentrate the DNA. Wondering if anyone has access to a Speed Vac?

- grace.yi