IGEM:MIT/2006/Notebook/2006-7-12

To do

1. Check biobrick sequence results against GenBank sequences
2. Make liquid cultures from YYC912 plates
3. Continue GC standards/readings work
4. Miniprep and digest/sequence (both) mutagenesis transformants
5. run a gel of and then biobrick the stationary phase promoter PCR product
6. Hook up each biobrick coding region (SAMT, BAMT, BSMT, ATF1) to a Rbs, Promotor, and Terminator

Biobrick Progress

Made a miniprep from the liquid culture made up last night carrying the terminator B0015. Also made a glycerol of the B0015 culture.

Did the following digest for later ligation between the SAMT coding region and B0015 and between the BSMT coding region and B0015 (the other coding regions are in the middle of testing after site-directed mutagenesis).

SAMT

30.2 uL H20

5 uL Buffer 2 (vortexed quickly)

.5 uL BSA

13.25 uL 60.4 ng/uL DNA

.5 uL EcoRI

.5 uL SpeI

BSMT

32.25 uL H20

5 uL Buffer 2 (vortexed quickly)

.5 uL BSA

10 uL 71.2 ng/uL DNA (ran out, ideal would have been 11.25 uL)

.5 uL EcoRI

.5 uL SpeI

B0015

39.5 uL H20

5 uL Buffer 2 (vortexed quickly)

.5 uL BSA

4 uL 203.9 ng/uL DNA

.5 uL EcoRI

.5 uL XbaI

• Note: We are looking for promoters already attached to RBSs, so we can skip that assembly process.

Coding Region/Terminator Ligation

• We Eco/Spe cut BSMT and SAMT and Eco/Xba cut B0015, did a PCR cleanup, ligated, and transformed.
  • Concentrations of cut products: BSMT- 13.3μg/L; SAMT- 10.1μg/L; B0015- 10.7μg/L

Prep for GC

Grew up liquid cultures of Dudareva's BSMT and SAMT in LB Kan as well as BL21 in LB as a control.

Mutagenesis Progress

There were no ATF1 colonies. There were 2 BAMT colonies. We made two liquid cultures from the 2 BAMT colonies.

osmY promoter

Ran a gel of the osmY promoter (both truncated and full regions) with controls. We did not see any bands. We plan on rerunning the PCR first thing tomorrow.

Email requests progress (kb)

1. Recieved a tnaA5 mutated e. coli strain from Yale, liquid culture growing tonight
   • Unfortunately, this particular mutation may not be 'tight-enough' to stop all indole activity
   • Mary at Yale recommends 2 other strains (first = an insertion mutation, 2nd = a deletion mutation) as other options to test.
   • I ordered these 2 strains today
2. Recieved SA biosynthesis pchBA pUC18-derivative plasmid from Switzerland today, transformants growing up on Amp plates tonight
   • Thank you to Coni Reimmann!
3. Requested methylobacterium shuttle vector pWUBR from different listed author (J. Hubacek)
4. Requested an E. coli S17-1 strain that harbors a plasmid for cloning into methylotrophs (T. Leisinger)
5. Waiting for reply from various other contacts