IGEM:MIT/2006/Notebook/2006-7-18

Transformants

1. IK control did not grow, though BSMT and BAMT grew
2. ATF1 and Top10 control grew as well
   • Next step is liquid culture all of them, and miniprep on ATF1
3. SAMTC connected to both RBSs grew along with SAMTA connected to both RBSs and the control.

5 mL Liquid Cultures Made

B0030.SAMTC.B0015 from colonies A, B, C, & D

B0032.SAMTC.B0015 from colonies A, B, C, & D

B0030.SAMTA.B0015 from 1 colony

B0032.SAMTA.B0015 from 1 colony

R0040

ATF1 Mutagenesis A & B

Dudareva's BAMT in the Indole Knockouts

Dudareva's BSMT in the Indole Knockouts

osmY Primer Design Part 2

Truncated Forward Primer: 5'- GTT TCT TCG AAT TCG CGG CCG CTT CTA GGCT TAT GTT TTC GCT GAT ATC CC - 3'

Total Length: 51 bp

Annealing Length: 23 bp

GC Content: 43.5%

Melting Temperature: 54.0 degrees C

hairpin deltaG: -2.66 kcal/mol

self dimer deltaG: -22.78 kcal/mol

Full Forward Primer: 5'- GTT TCT TCG AAT TCG CGG CCG CTT CTA GCTG GCA CAG GAA CGT TAT C - 3'

Total Length: 47 bp

Annealing Length: 19 bp

GC Content: 52.6%

Melting Temperature: 53.4 degrees C

hairpin deltaG: -3.99 kcal/mol

self dimer deltaG: -22.78 kcal/mol

Reverse Primer: 5'-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TGT TAA ATA TAG ATC ACA ATT TTG AAA CCG- 3'

Total Length: 60 bp

Annealing Length: 31 bp

GC Content: 25.8%

Melting Temperature: 53.5 degrees C

hairpin deltaG: -2.86 kcal/mol

self dimer deltaG: -25.98 kcal/mol

heterodimer deltaG with Forward Primer: -25.98 kcal/mol

heterodimer deltaG with Conserved Forward Primer: -25.98 kcal/mol

Meeting

• Tomorrow: Sequence: 4 SAMT-C connected to rbs & term-30, and 4 SAMT-C connected to rbs & term-32
• end of the day: Liquid culture all transformants (Duda. BAMT in indole-knockout, Duda. BSMT in indole-knockout, the two above)
• Make liquid culture from the glycerols of the constitutive promoters
• Veena: miniprep pCHBA, sequence it with primers from Reshma
• Andre- request plasmid for vanilla
• maybe: re-cut miniprepped BSMT
• research exponential phase promoter

Additional Smells

1. Myrcene and Linalool have floral scents
2. Myrcene synthase: http://content.febsjournal.org/cgi/content/full/268/21/5633

Annealing Parts for Synthesis of the osmY Promoter

Forward Strand:

5'-ctagaggcttatgttttcgctgatatcccgagcggtttcaaaattgtgatctatatttaacaaatactagtagcggccgctgca-3'

Length: 84 bases

GC Content: 41.7%

Melting Temperature: 69.7 degrees Celsius

Complementary Strand:

5'-GCGGCCGCTACTAGTATTTGTTAAATATAGATCACAATTTTGAAACCGCTCGGGATATCAGCGAAAACATAAGCCT-3'

Length: 76 bases

GC Content: 40.8%

Melting Temperature: 69.1 degrees Celsius

Heterodimer DeltaG: -161.93 kcal/mol

Also, I contacted Professor Schellhorn last night inquiring about a plasmid containing osmY or the sequences of the primers he used to PCR osmY out of E. coli.