Team:Alberta/Notebook

May.9-11.2012

Rick and Tom were recruited and started learning basic laboratory protocols, such as making competent cells, restriction digests, cell transformation, gel electrophoresis, ect.

May. 24

Rick, Tom, Spencer, and Easwar learned how to use PCR today, with varying degrees of success.

May 31

Today Tom finally managed to get a PCR to work, though it took him about twelve attempts.

June 4

Made working stock of puc19 and then transformed Top10 with puc19. Overnight cultures were also set up in preparation for the making of competent cells

June 5

Competent cells were made today using a standard procedure which took the entire day. The competent cell procedure is described in this wiki.

June 6

The competent cells were tested with basic puc19 transformation, and the transformation worked.

June 7-8

Today multiple PCRs were run on puc19 with two of the color genes and a repressor gene.

June 11

Made pUC19 plasmid with the new color genes and C1 and transformed it into cells.

June 18-21

We cloned a variety of different promoters (nine total) into our color gene plasmids in order to get a sense of relative promoter strengths. we also seqencing those construct to check they are correct.

June 22- July 3

We used PCR to add new RBS colour genes and regulatory promoters to our constructs. We then pieced those constructs into kanamycin resistant plasmid backbone and transform them into Top 10 cells.

July 4-5

Torrin and Sarah join. we perform the same work again on TG1.

July 6

Tom checked overnights of origin cut site strains and chose colonies to be sequenced.

July 9-10

Today Tom Used the successful origin cut site plasmid with Amp Resistance and replaced that resistance with Chlr. Torrin and Sarah are creating and testing chemical gradient plates, and learning additional laboratory procedure. Rick and Spencer is making competent cells of TG1 so that we can test our current parts in an E.Coli strain that grows faster than our current Top10.
Rick and Easwar are drop in LacI and Tet R repressor into chloranphenicol construct.

July 11

We continued our experiments with making chemical gradients on agar plates.

July 12-17

we successfully get the LacI and TetR repressors into chlr contstruct and co-transfrom with RTa and RL we created from july 7. the result showed seemed it is working and we have white colony.Then we decide to send TetR and LacI construct for sequencing. The result showed LacI and TetR sequence were good.

July 18

The Lac and Tet promoter pieces have been successfully added to plasmids via specially designed PCR primers, and will control the origin of replication on said plasmids. We will start testing them over the next couple of weeks.

July 19

we decide to send TetR and LacI construct for sequencing. The result showed LacI and TetR sequence were good.

July 20-25

co-transformation of lacI/tetR promoter with red gene and repressor (lacI and TetR) gene were successfully, however, when we do the reverse experiment (LacI/tetR with non-repressor gene) with isolated white colonies were not many red colonies.

July 30- Aug 13

Tom on vancation. Rick take over Tom’s work continuing on the research on the process of controlling plasmid copy number in the cell. we redesign the plasmid so that Tom’s original desing wont killing cells by introducing lac operator or tet operator into the wild ori promotor with NsiI site beside it.