IGEM:MIT/2006/Notebook/2006-7-10

Sequencing

 * Refer here for sequencing protocol.
 * We already have the necessary primers (VR1 and VF2).
 * Note: You only set up the sequencing reaction with one of these two primers
 * Add the following volumes for a 10-reaction master-mix:
 * 90&mu;L H2O
 * 1&mu;L primer
 * Add 9.01&mu;L to each tube
 * Add 3&mu;L respective DNA

Reaction Wells:

1. SAMT-A VF2

2. SAMT-B VF2

3. SAMT-C VF2

4. SAMT-D VF2

5. BSMT-A VF2

6. BSMT-C VF2

7. BSMT-E VF2

8. BAMT VF2

9. SAMT-A VR

10. SAMT-B VR

11. SAMT-C VR

12. SAMT-D VR

13. BSMT-A VR

14. BSMT-C VR

15. BSMT-E VR

16. BAMT VR

17. ATF1 VF2

18. ATF1 VR

osmY Promoter
I had the primers for the osmY (stationary phase promoter) ordered after having them checked by Barry. osmY is naturally in E. coli; thus, the E. coli genome will be used as a template in the PCR process. The plan is to biobrick the part after PCR and then connect it to SAMT and/or BSMT, which have been consistently smelly. The osmY promoter has been reported by Schellhorn, et al. to show approximately 50-fold more B-galactosidase activity in the stationary phase than in the exponential phase when connected to the LacZ gene. This promoter is activated by rpos, the gene product which is attached to Francois's stationary phase promoter. Thus, it seems that this promoter will have a much sharper contrast in expression depending on the cell phase than Francois's promoter would have. Preliminary fluorescence tests were done with Francois's stationary phase promoter on the plate reader and a control plasmid but the results showed only a slight increase in expression of GFP by Francois's promoter in the stationary phase.

Expressing the Biobricked Coding Regions with Other Biological Parts
We need to connect the hopefully successfully biobricked enzyme coding regions to promoters, RBSs, and terminators from the Registry. We were planning on testing one promoter, two RBSs, and one terminator. They are listed below:

Promoters:

R0040

RBSs:

B0030

B0032

Terminator:

B0015

To do this, we plan on digesting each of the plasmids and ligating them appropriately, first connecting the promoters to the RBSs and the coding regions to the terminators. Then, we would connect the two intermediate pieces together to create a cassette. We could not find promoters already attached to RBSs in the Registry. All we could find were cassettes with other coding regions already inserted.

Isoamyl alcohol primers
2-step process...need BAT2 and THI3 from yeast.

BAT2: 1 atgaccttgg cacccctaga cgcctccaaa gttaagataa ctaccacaca acatgcatct 61 aagccaaaac cgaacagtga gttagtgttt ggcaagagct tcacggacca catgttaact 121 gcggaatgga cagctgaaaa agggtggggt accccagaga ttaaacctta tcaaaatctg 181 tctttagacc cttccgcggt ggttttccat tatgcttttg agctattcga agggatgaag 241 gcttacagaa cggtggacaa caaaattaca atgtttcgtc cagatatgaa tatgaagcgc 301 atgaataagt ctgctcagag aatctgtttg ccaacgttcg acccagaaga gttgattacc 361 ctaattggga aactgatcca gcaagataag tgcttagttc ctgaaggaaa aggttactct 421 ttatatatca ggcctacatt aatcggcact acggccggtt taggggtttc cacgcctgat 481 agagccttgc tatatgtcat ttgctgccct gtgggtcctt attacaaaac tggatttaag 541 gcggtcagac tggaagccac tgattatgcc acaagagctt ggccaggagg ctgtggtgac 601 aagaaactag gtgcaaacta cgccccctgc gtcctgccac aattgcaagc tgcttcaagg 661 ggttaccaac aaaatttatg gctatttggt ccaaataaca acattactga agtcggcacc 721 atgaatgctt ttttcgtgtt taaagatagt aaaacgggca agaaggaact agttactgct 781 ccactagacg gtaccatttt ggaaggtgtt actagggatt ccattttaaa tcttgctaaa 841 gaaagactcg aaccaagtga atggaccatt agtgaacgct acttcactat aggcgaagtt 901 actgagagat ccaagaacgg tgaactactt gaagcctttg gttctggtac tgctgcgatt 961 gtttctccca ttaaggaaat cggctggaaa ggcgaacaaa ttaatattcc gttgttgccc 1021 ggcgaacaaa ccggtccatt ggccaaagaa gttgcacaat ggattaatgg aatccaatat 1081 ggcgagactg agcatggcaa ttggtcaagg gttgttactg atttgaactg a


 * Forward BAT2 primer: 5'- GTT TCT TCG AAT TCG CGG CCG CTT CTA Gat gac ctt ggc acc cct aga cg -3'
 * Reverse BAT2 primer: 5'- GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TAT TAg ttc aaa tca gta aca acc -3'

THI3: 1 atgaattcta gctatacaca gagatatgca ctgccgaagt gtatagcaat atcagattat 61 cttttccatc ggctcaacca gctgaacata cataccatat ttggactctc cggagaattt 121 agcatgccgt tgctggataa actatacaac attccgaact tacgatgggc cggtaattct 181 aatgagttaa atgctgccta cgcagcagat ggatactcac gactaaaagg cttgggatgt 241 ctcataacaa cctttggtgt aggcgaatta tcggcaatca atggcgtggc cggatcttac 301 gctgaacatg taggaatact tcacatagtg ggtatgccgc caacaagtgc acaaacgaaa 361 caactactac tgcatcatac tctgggcaat ggtgatttca cggtatttca tagaatagcc 421 agtgatgtag catgctatac aacattgatt attgactctg aattatgtgc cgacgaagtc 481 gataagtgca tcaaaaaggc ttggatagaa cagaggccag tatacatggg catgcctgtc 541 aaccaggtaa atctcccgat tgaatcagca aggcttaata cacctctgga tttacaattg 601 cataaaaacg acccagacgt agagaaagaa gttatttctc gaatattgag ttttatatac 661 aaaagccaga atccggcaat catcgtagat gcatgtacta gtcgacagaa tttaatcgag 721 gagactaaag agctttgtaa taggcttaaa tttccagttt ttgttacacc tatgggtaag 781 ggtacagtaa acgaaacaga cccgcaattt gggggcgtat tcacgggctc gatatcagcc 841 ccagaagtaa gagaagtagt tgattttgcc gattttatca tcgtcattgg ttgcatgctc 901 tccgaattca gcacgtcaac tttccacttc caatataaaa ctaagaattg tgcgctacta 961 tattctacat ctgtgaaatt gaaaaatgcc acatatcctg acttgagcat taaattacta 1021 ctacagaaaa tattagcaaa tcttgatgaa tctaaactgt cttaccaacc aagcgaacaa 1081 cccagtatga tggttccaag accttaccca gcaggaaatg tcctcttgag acaagaatgg 1141 gtctggaatg aaatatccca ttggttccaa ccaggtgaca taatcataac agaaactggt 1201 gcttctgcat ttggagttaa ccagaccaga tttccggtaa atacactagg tatttcgcaa 1261 gctctttggg gatctgtcgg atatacaatg ggggcgtgtc ttggggcaga atttgctgtt 1321 caagagataa acaaggataa attccccgca actaaacata gagttattct gtttatgggt 1381 gacggtgctt tccaattgac agttcaagaa ttatccacaa ttgttaagtg gggattgaca 1441 ccttatattt ttgtgatgaa taaccaaggt tactctgtgg acaggttttt gcatcacagg 1501 tcagatgcta gttattacga tatccaacct tggaactact tgggattatt gcgagtattt 1561 ggttgcacga actacgaaac gaaaaaaatt attactgttg gagaattcag atccatgatc 1621 agtgacccaa actttgcgac caatgacaaa attcggatga tagagattat gctaccacca 1681 agggatgttc cacaggctct gcttgacagg tgggtggtag aaaaagaaca gagcaaacaa 1741 gtgcaagagg agaacgaaaa ttctagcgca gtaaatacgc caactccaga attccaacca 1801 cttctaaaaa aaaatcaagt tggatactga


 * forward THI3 primer: 5'- GTT TCT TCG AAT TCG CGG CCG CTT CTA Gat gaa ttc tag cta tac aca gag -3'
 * reverse THI3 primer long: 5'- GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TAT TAg tat cca act tga ttt ttt ttt aga ag -3'

GC progress

 * We figured out how to use the GC and ran 3 concentrations of methyl salicylate to determine the order of magnitude of the concentration we should add. A dilution of 1:10000 was best.  We also found that the methyl salicylate came out at around 9.05 minutes.
 * So far, we've run the following samples: BSMT induced, BSMT un-induced, SAMT induced, and SAMT un-induced.
 * results: we don't have an internal standard yet so we can't quantify our restults (pentachloronitrobenzene is coming on weds, hopefully, so we can re-do these runs and quantify how much methyl salicylate is present in our samples). Qualitatively, it seemed like there was much more methyl salicylate in the BSMT+ and SAMT+ than there was in BSMT- and SAMT-.  Also, there was a strange molecule with MW 117 that showed a peak at 10 minutes in the BSMT-, SAMT+, and SAMT- fractions...we're not sure what this is.  There were no other big peaks, so we think that this 117 peak might be SA (?)
 * We need to run more controls!!! Haven't had the GC time to do this yet...but in the very near future, we will!