Team:Amsterdam/Notebook/syn dependence

iGEM Amsterdam 2015

Lab Notebook for making Synechocystis dependent on E. coli

Week 1 May 3-10
Week 1 May 11-17

Week 2 May 18-24

Week 3 May 25-31
Planned to Knock out Arginine in Syn
Week 4 June 1-7
Designed Primers
Week 5 June 8-14
Primers arrived, learned about the lab
Week 6 June 15-21
Conducted transformation of PFL-SA plasmid + the up/down homologous regions of ArgH gene. Used LB/agar media + Ampicilin plates. Left Overnight. Conducted colony PCR on the 12 colonies using the forward/reverse primers of the ArgH homologous region insert. All 12 colonies showed activity around 2k (size of homologous fragment). Replated colonies to a fresh plate. Inoculated colony 2 for future extraction Extracted plasmid from colony 2. Saved some of inoculant in -80 freezer. Conducted PCR on the extracted plasmid to verify presence of insert. Conduct digestion on both PFLSA + homologous region plasmid and the Pwd plasmid with nickel insert. PFLSA plasmid digestion had strange bands. PwD plasmid looked okay so extracted the top band. using gel extraction Next Week : Figure out what happened with PFLSA plasmid. Need to insert the PWD insert into pflsa plasmid and transform to get the final knock out plasmid to insert into syn
Week 7 June 22-28
Repeated digestion at a smaller scale and found that the insert might not have been in that particular plasmid. So just extracted plasmid from other colonies. Other colonies gave the proper results in the mini digestion check Chose the colony that gave the best mini digestion report and digested all of it then PCR purified product Did ligation of the PFSLA up/down with the insert. Left at room temp for 30 min then did an overnight ligation Transformed the final ligated product into E. coli for overnight transformation Led to many colonies . Did colony PCR and two of the 12 picked colonies looked promising. Next Week: Extract plasmid and transform synechocystis
Week 8 June-July 29-5
Inoculated colonies that were promising in colony PCR overnight. Extracted the plasmids and did digestion check for both the colony’s extracted plasmids For gels Both had proper bands after digestion and proper bands for the final construct Started transformation of Synechocystis according to Wei’s protocol. Was given some WT syn by Wei which was washed, spun down and reconstituted in a more concentrated amount. The final construct was added and left to shake in light for 5 hours. After this, the cells were spread on a membrane on top of BG11 plate without Antibiotics to grow overnight The next day the cells were transferred to a fresh plate with antibiotics (Kanamycin). This will grow for 6/7 days in light Next Week: Conduct sequencing for the original PFSLA up/down plasmid to verify we got the right plasmid. Pick colonies from the membrane with syn cells on it to a fresh BG11/Kanamycin plate
Week 9 July 6-12
Put the PFSLA + up/down of ArgH plasmid up for sequencing AFter cells in Syn membrane grew transferred some colonies into separate BG11 plates including Kanamycin. Will probably repeat transformation as these cells should theoretically need Arginine in the BG11 which has none. Next Week: Make BG11 + Arginine Plates
Week 10 July 13-19
Made MANY BG11 plates. Made just BG11, BG11 + Kanamycin + Arginine, BG11 + Kanamycin, and BG11 + kanamycin + arginine Repeated transformation of Synechocystis with fresh construct from a different colony. This time transformed both wild type synechocystis as well as the overproducing ACS mutant synechocystis. First step used just BG11 plates without any extra ingredient to grow overnight After overnight growth, the membrane was transferred into BG11 plates containing Kanamycin and Arginine. Will need to grow for 6/7 days Primers for Proline knock out arrived. Started the process to knock out proline. Conducted PCR of up/down stream region of the Proline gene. But didn’t see any signal from the gel. Might have been because of improper genomic DNA Next Week: Use a different genomic DNA for amplification of up/down region
Week 11 July 20-26
Since last weeks Syn transformation took most of the final construct. Inoculated both colonies contianing the final construct overnight. Final construct was extracted from the colonies Redid the PCR for the up/down region with different genomic DNA. The down region was able to be amplified. But the up region did not. Extracted the down fragment. Multiple repeats of trying to amplify the up region with different annealing temperatures. (going all the way down to 30). Nothing worked. Switched to mytaq Transferred the syn colonies of the ArgH knockout from last weeks membrane into a fresh BG11 plate contianing Arginine and Kanamycin. Will again need to grow for 6/7 days Some of the sequencing reactions failed. The PFSLA + up.down plasmid for ArgH was depleted so had to inoculate from the colony, extract and produce more plasmid inorder to do sequencing. Sent in the plasmid for sequencing Next Week: Figure out what is going on with the Proline upstream gene.
Week 12 July-August 27-2
In order to troubleshoot why I was having diffuclty amplifying the upstream region of the Proline gene, decided to throw away the primers I was using and made a fresh aliquot. Conducted PCR again with 50C and 40C annealing temp. 40C had weird result and 50C had nothing. Repeated with even lower annealing temperatures (30 and 40) however increased the amount of cycles to 35. Again the results were not as clean, there was many nonspecific binding. But at least there was something. Conducted a gradient of annealing temperatures using 35 cycles from temp 33 - 50. This time the results seemed a bit more clear with definite bands around 1kb. The 50C band looked the best.
Week 13 August 3-9
Conducted a gradient PCR for proline at 5 different temperatures using TAQ. Was able to amplify from 35-50 C Repeated with Hercules ended in failure Conducted colony PCR of Syn. for arginine. Unforatunately used the wrong protocol so will have to repeat again. For upstream repeated with both herc and taq polymerase of Arginine. Hercules didn't work whereas Taq showed many good bands. Just went on and extracted the proline upstream from taq
Week 14 August 10-16
Fused the proline up amplified by Taq with the down amplified by herc. Did amplication of fusion gel proved negative Redid colony pcr of arginine ko colonies. none of the colonies had the cassette we are expecting at 5.7kb Did col PCR of the first time I tried to transform where there was no arginine. Again the gel proved negative for the mazF cassettte Tried transforming syn again. Transforming ACS, WT, and a WT control. Doing this to make sure as a negative control the Kan works. Tried different polymerases for Proline knock out. Herc, Taq and PFU. Only taq worked
Week 15 August 17-23
Checked the colonies transformed 3 weeks ago for single crossover. Used different primers Uprev and down forward. Made more of construct to transform syn with. The single cross over check proved negative. Made half a liter of bg 11. Redid col PCR checked with diff primers to make sure the casette was in. Also included a plasmid control. THe casette was definitely in. Not sure why gene wasn't knocked out. Tried proline upstream amp for the final time. This time i included a dmso gradient. This again failed so just went on and did TAQ again with hope of fusing later. Tried to fuse the proline fragments and this failed.
Week 16 August 24-30
Week 17 August-September 31-6

Week 18 September 7-13

Week 18.5 September 14-18