Difference between revisions of "Team:Uppsala/Notebook"
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- 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.<br> | - 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.<br> | ||
- 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.<br> | - 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.<br> | ||
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- 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.<br> | - 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.<br> | ||
- 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.<br> | - 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.<br> | ||
Revision as of 15:17, 31 October 2017
Week: 07/06-16/06
- A safety demonstration was done by the Lab technicians and Project managers which enabled us to know the fire escapes, first aid and emergency contacts. Lab space was assigned.
- Buffers and stock solutions were prepared. LB media and LB-agar plates with different antibiotics (Kanamycin, Chloramphenicol and Tetracycline) were prepared.
- E. coli DH5α competent cells were prepared. Ordered constructs for the three genes (CCD, ADH and UGT) were digested with available enzyme (E and S) and ligated to Linearized plasmid backbones from the iGEM Kit.
- Prepared competent cells were tested with the iGEM Competent Cells Test kit. No growth was observed. Possible causes: Faulty competent cells, improper digestion or transformation protocol.
- Made DH5α competent cells with another new protocol to be tested and used later.
Week: 19/06- 22/06
- Testing of the competent cells made last week was done with the competent cells test kit. There was growth suggesting that competent cells were working good.
- To test if the restriction digestion, ligation and transformation worked well, they were repeated again with the newly prepared and tested competent cells above. No growth was observed even when high concentrations were plated, suggesting that the fault was in the Restriction digestion, ligation steps
Week: 26/06-30/06
- On expert suggestion, it was understood that the enzymes need few extra bases on the ends for efficient activity. Thus we designed and ordered primers that would add overhangs to our constructs. Other reason could be that the promoters were too strong and the genes were toxic to the cell. So a set of primers were also designed to remove promoter from between the prefix and RBS.
- To circumvent the high toxicity of the gene, we decided to clone the devices in low copy plasmids. For that low copy plasmids were identified from the master strain database of glycerol stock at the lab, grown overnight in agar plates, grown overnight in LB and plasmids were purified.
- Digestion was tested with X and S to optimize the protocol for digestion
Week: 03/07-7/07
- The primers designed last week were tested and PCR Parameters optimized. PCR amplicons were electrophoresed and positive sections were eluted from the gel and their concentrations measured.
- Low copy plasmid backbones purified last week were digested and linearized. The PCR amplified constructs were digested and ligated to Low copy plasmid backbone. Transformation was done
- High copy plasmid backbones from the glycerol stock were purified.
Lambda red integration:
- Planning/research for project.
- Made competent cells of IS150 and gsp strain.
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.
- 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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