IGEM:MIT/2006/Notebook/2006-8-15
Overall Progress
WGD
Completed with B0030 and B0032 and connected to R0040. In the process of connecting it to R0011. All intermediates are in the -80 degree freezer as glycerols.
BSGD
Completed with B0030 and B0032 and connected to R0040. In the process of connecting it to R0011. All intermediates are in the -80 degree freezer as glycerols.
SAGD
We connected pchBA to a terminator yesterday. We will receive sequence confirmation on Friday. The part will be mutagenized Wednesday after making a liquid culture tonight. Then, we can connect it to an RBS and a promoter. Primers for PCRing another SAGD coding region (pmsCEAB) out of plasmid pE3 arrived today. pE3 should arrive this week.
IAOHGD
We connected B0030.BAT2 to B0030 yesterday. BAT2 is mutagenized after gel confirmation of a digest. We will receive sequence confirmation on this piece on Friday. We are currently running colony PCR to see if THI3 was successfully placed into a vector at the SpeI and XbaI sites after using antarctic phosphatase step. If it was successfully placed into the vector, we plan to use site-directed mutagenesis to eradicate the PstI site in THI3. Then, we can connect it to a terminator and eventually connect it to the B0030.BAT2.B0030 part.
osmY Promoter
We just cannot get it to ligate to anything. Many assemblies with E0840 (the RBS.GFP.Terminator part) failed along with assemblies with RBS.ATF1.Terminators and RBS.BSMT.Terminators. We are currently starting from the top. We are rePCRing the pieces out of the E. coli genome and reannealing the primers containing osmY.
Inverters
Tomorrow, we should have sequencing for the ligations of the inverter Q04400 to the RBS.coding.terminators of both the BSGD and WGD.
Pseudomonas
Salicylic Acid generatoring strains of the organisms in the mail. Research being done on the type of media required for the strain to grow. We will receive sequence confirmation of the R0040.WGD in the shuttle vector tomorrow.
Device Characterization of BSGD and WGD
We plan to do a time course at varying ODs and varying concentrations of precursors for R0040.B0030.coding.B0015, R0040.B0032.coding.B0015, R0011.B0030.coding.B0015, and R0011.B0032.coding.B0015 for both the BSGD and WGD. We plan to set up the time course experiment using our standard precursor concentrations tomorrow (just for the R0040 parts) and measure the culture's approximate end product concentration with the GC Thursday.
Groups
We have divided the project into very rough miniprojects:
GC- Veena, Andre
IAOHGD- Bo, Stephen
osmY- Andre
Pseudomonas- Kate
Registry Entries- Bo
SAGD- Kate, Veena, Stephen
Digest
Two digests of R0011 cut with E and S were made.
Plan to do a three-part assembly with the R0011 and the X and P cut RBS.coding.terms for both the wintergreen and banana devices.
We will later use this to see which mixed and matched promoters yield the most favorable GC results.
Transformants Results
Growth on the following plates:
pUC18
pCHBA (AMP) B.B0015
pCHBA (A/K) A.B0015
pCHBA (A/K) C.B0015
30.BAT2 C Mut A.30
THI3 in pSB104.1
THI3 iin pSB104.1 (antarctic phosphatase)
THI3 in pSB104.1 (red top/antarctic phosphatase)
THI3 in pSB104.1 (red top)
No growth on the following plates:
osmY.E0840
30.BAT2 C Mut A.32
THI3 colony PCR
- keep: E, H, J, K
