Difference between revisions of "Team:Uppsala/Notebook"

Week: 10/07-14/07/h3>

- Low copy plasmids transformed last week were re-streaked. Colony PCR followed by Gel electrophoresis was done on them.
- Positive colonies, screened from colony PCR were re-streaked and glycerol stock was made for them.
- Electroporation-competent DH5α cells were prepared.
- The PCR amplified constructs were restricted digested and ligated to high copy plasmid backbones cells. These plasmids were then transformed by heat-shock.
- More low copy plasmid for each antibiotics with Red fluorescent protein RFP were purified from the master stock.
- UGTCs2 gene was cloned in pSB1C3 plasmid backbone since using tetracyclin was being troublesome.
- Electro-competent cells were tested with cloned genes. No growth was observed. Possible cause: cells died during electroporation. Desalt the ligated DNA.
- Colony PCR of the positive colonies (white colonies) obtained from heat shock transformation of cloned genes in high copy plasmids. No bands visible..

Lambda red integration:

- Tested transduction with bgl strain as the donor and MG1655 as receiver (Cat-SacB will be transduced). 1 colony was found on the plate with chloramphenicol. This one was restreaked on chloramphenicol which then had more growth.
- Tried to insert CrtI gene in IS150 strain and dhfr gene in gsp strain using lambda red. The IS150 strain grew on sucrose (indicating that the SacB sequence had been replaced) but tetracycline had not been added so the strain could have lost the lambda red plasmid. The gsp strain had strange growth pattern.

Week: 17/07-21/07

- Fresh start of Restriction digestion and Ligation of PCR amplified constructs into high copy plasmids. Used both Heat shock (with commercial competent cells) and Electroporation.
- Positive (white) colonies were restreaked and colony PCR was done. No bands were observed.
- Troubleshooting of colony PCR. Optimised PCR parameters and new buffer components.
- SDS-PAGE parameters were optimised and a test run was done with BSA dilutions. No bands were observed in the first run due to improper buffer. Troubleshoot and Run again!

Lambda red integration:

- PCR of inserts CrtI, dhfr, CrtZY and CrtEB. Gel elecrophoresis for verification (see figure below).
- PCR of inserts CrtZY and CrtEB. Gel electrophoresis for verification but there was nothing on gel Purification of inserts CrtI and dhfr.
- Gel electrophoresis of pooled PCR products of insert CrtEB and CrtZY from 17/7 (see figure below). Electroporation of purified CrtI gene in IS150 strain and purified dhfr gene in gsp strain. Put overnight on shaking water bath.
- The overnight cultures of IS150 strain was streaked on tetracycline and sucrose plates and gsp strain was streaked on tetracycline and trimetoprim plates. Gel electrophoresis of CrtEB insert (see figure below).

Week: 24/07-28/07

- Positive colonies carrying construct in tetracycline backbone from last week were re-streaked to check for viability.
- Buffers for immobilised metal affinity chromatography (IMAC) were prepared and degassed. IMAC column was packed.
- Positive cells (white colonies) from containing genes in high copy plasmids were grown overnight and sonicated. The lysates were purified by IMAC.
- More LB agar plates with antibiotics were prepared.

Lambda red integration:

- PCR of insert CrtEB. Restreak clones of IS150 strain from 21/7 on sucrose and tetracycline plates and gsp strain from 21/7 on and tetracycline and trimetoprim plates. Liquid cultures with same respective antibiotics for the strains were made.
- Gel electrophoresis of CrtEB PCR product from 24/7 (see figure 4). Restreak colonies of IS150 strain from 24/7 plates on sucrose plates then chloramphenicol plates. Prepare cultures for transduction of IS150 by growing overnight in P1 LB. Purify CrtZY and CrtEB and use nano drop on all purified inserts to check DNA concentration (see table 1). Ordered new primers for CrtEB.
- gsp strain did not grow on plates but did in liquid culture. 5 of 8 colonies of IS150 strain from 25/7 grew on sucrose but not chloramphenicol (see figure 5 & 6). New plates were made (tet + chlor, tet + tmp, tet + suc, chlor). Transduction of bgl strain as the donor and IS150 as receiver. Take some gsp liquid culture and put in some tet + sucrose to make sure it’s our strain that’s growing. Make liquid cultures with gsp (dhfr) in tet + tmp with the different tet to check if antibiotics work.
- No growth on transducer IS150 from 26/7. Start overnight culture of bgl strain to make lysate (donor strain). Colony PCR of the colonies from 26/7 that grew on sucrose but not on chlor and gel electrophoresis to check inserts. Make tet + chlor and tet + tmp plates. Start overnight cultures of IS150 strains with inserts that were confirmed by colony PCR.
- Colony PCR of gsp strain and gel electrophoresis
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