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Week 1: 25th - 31st
25th July | Eloise & Ezekiel
- Plasmid PCR of the pet19b backbone to prepare for Gibson Assembly.
26th July | Div, Eloise & Ezekiel
- Visualised PCR from day prior on an agarose gel using gel electrophoresis.
27th July | Div, Eloise & Ezekiel
- Repeated PCR with different conditions; ran another gel.
29th July | Div, Eloise & Ezekiel
- Troubleshooted primers for pet19b amplification.
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Week 2: 1st - 7th
2nd August | Div, Eloise & Ezekiel
- Plasmid PCR and Gel Visualisation.
- DNA for 4 fungal proteins arrived and were prepared.
3rd August | Div, Eloise & Ezekiel
- Gibson assembly and transformation of PstSCR1, PstHXT1, Pgt_IaaM and Pst_12806 into NEB TURBO E.coli.
4th August | Div & Ezekiel
- Checking plates and streaking.
5th August | Div & Ezekiel
- cPCR for SCR1 and IaaM and ran DNA gel electrophoresis.
Week 3: 8th - 14th
8th August | Div & Ezekiel
- Plate Streaking.
- Prepared overnight culture for the colonies used in the cPCR on the 5th.
9th August | Div, Eloise & Ezekiel
- Miniprep of 4 promising SCR1 colonies from overnight cultures.
12th August | Eloise & Ezekiel
- Repeated cPCR for SCR1 and IaaM.
Week 4: 15th - 21st
15th August | Eloise & Ezekiel
- Pet19b digest that was forgotten in the incubator (El & Ez).
16th August | Eloise & Ezekiel
- Miniprep of colonies from cPCR on 12th August.
- 3 Colonies of SCR1 in NEB TURBO E.coli were prepared and sent to for Ramaciotti Centre of Genomics for Sequencing.
- Miniprep of pet19b backbone.
- Gibson Assembly for HXT1 and Pst_12806.
19th August | Div, Eloise & Ezekiel
- Transformed SCR1 into NEB T7 Express E.coli.
- Transformed HXT1 and Pst_12806 into NEB TUBRO E.coli.
Week 5: 22nd - 28th
22nd August | Div, Eloise & Ezekiel
- Performed cPCR of Pst_12806 (6 colonies) and PstHXT1 (6 colonies) in NEB TUBRO E.coli.
23rd August | Eloise & Ezekiel
- Repeated cPCR of Pst_12806 (6 colonies) and PsTHXT1 (6 colonies) in NEB TUBRO E.coli with different colonies.
25th August | Eloise & Ezekiel
- Set up overnight cultures for protein expression of PstSCR1.
26th August | Div, Eloise & Ezekiel
- Day culture and harvest for protein expression of PstSCR1.
- Pet19b gel extraction.
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Week 6: 29th August - 4th September
29th August | Eloise & Ezekiel
- Gibson assembly & transofrmation of Pst_12806 and PstHXT1 into NEB TURBO E.coli.
- Prepared for PstSCR1 protein expression optimization on the following day.
- Performed protein expression check (Bugbuster) for PstSCR1 day culture from the 26th of August.
30th August | Eloise, Ezekiel & Roger
- Lysed cells by sonication and purified PstSCR1 protein with gravity columns.
- Set up overnight culture of PstSCR1 in NEB T7 Express E.coli for protein expression optimisation.
31st August | Div, Ezekiel & Ryan
- Performed cPCR on transformed sampled from 29th August; results showed unsuccessful Gibson Assembly.
- Performed day cultures on PstSCR1 NEB T7 Express E.coli for protein expression optimization at different IPTG concentrations and temperatures.
- Harvested the PstSCR1 NEB T7 Express E.coli cultures that were incubated at 30℃ and 37℃ during the day culture; 16℃ culture was incubated overnight.
1st September | Div, Roger & Ryan
- Harvested the 16℃ culture PstSCR1 NEB T7 Express E.coli for protein expression optimization at different IPTG concentrations and temperatures.
- Performed cPCR on PstHXT1 and Pst_12806 transformations from the29th August.
2nd September | Div, Eloise & Ezekiel
- Performed protein expression check (Bugbuster) for PstSCR1 day culture from the 26th - 27th of August.
Week 7: 5th - 11th
5th September | Div & Ezekiel
- Restriction digest of Pet19b.
- Gibson assembly and transformation of Pst_12806 into NEB TURBO E.coli.
8th September | Div & Ezekiel
- Set up 2 overnight cultures of Pst_12806 in NEB TURBO E.coli.
9th September | Div, Eloise & Ryan
- 2 samples of Pst_12806 in NEB TURBO E.coli were prepared and sent to Ramaciotti Centre of Genomics for sequencing.
Week 8: 12th - 18th
12th September | Eloise & Ezekiel
- Set up overnight culture of PstSCR1 in NEB T7 Express E.coli in optimal conditions (inferred from previous protein optimisation).
13th September | Eloise, Roger & Ryan
- Performed day culture and harvest on PstSCR1 NEB T7 Express E.coli for protein expression.
14th September | Div & Ezekiel
- Performed protein expression check (Bugbuster) for PstSCR1 day culture from the 13th of September.
- Arrival of TaISP and SERK3B gBlocks were prepared and concentration was measured on NanoDrop.
15th September | Div, Eloise, Ezekiel & Ryan
- Gibson assembly and transformation of TaISP and SERK3B into NEB TURBO E.coli.
16th September | Eloise & Ezekiel
- cPCR of TaISP and SERK3B transformation from the 15th of Septmeber and Gel visualisation.
Week 9: 19th - 25th
19th September | Div, Eloise & Ezekiel
- Training on AKTA Start for purification of mCerulean and mVenus.
20th September | Eloise, Ezekiel, Roger & Ryan
- Training on AKTA Start for purification of mCerulean and mVenus.
- Ran SDS-gel for prufication on 19th September.
- Repeated cPCR of TaISP and SERK3B transformation from 15th September and Gel visualisation.
21st September | Div, Roger, & Ryan
- Ran SDS-gel for purification on 20th September.
- Repeated cPCR of TaIS and SERK3B transformation from 15th September and Gel visualisation.
22nd September | Div & Ezekiel
- Repeated Gibson Assembly and transformation of TaISP and SERK3B into NEB TURBO E.coli.
- Transformed Pst_12806 into NEB T7 Express E.coli.
23rd September | Div, Eloise, Ezekiel & Roger
- cPCR of TaISP and SERK3B transformation from 22nd September and Gel visualisation.
Week 10: 26th September - 2nd October
26th September | Eloise & Ezekiel
- Repeated cPCR of TaISP and SERK3B transformation from 22nd September and Gel visualisation; observed band of correct bp.
- Set up overnight culture of Pst_12806 in NEB T7 Express E.coli for protein expression.
27th September | Div, Eloise, Ezekiel & Roger
- Ran DNA gel electrophoresis for cPCR on the 26th of September.
- Preformed day culture and harvest on Pst_12806 NEB T7 Express E.coli for protein expression.
28th September | Div, Roger & Ryan
- Restreaked colonies for SERK3B in NEB TURBO E.coli.
- Reran DNA gel electrophoresis for cPCR on 26th September.
29th September | Div, Ezekiel & Ryan
- Set up overnight culture of TaISP in NEB TURBO E.coli for miniprep.
30th September | Eloise
- Miniprep of TaISP in NEB TURBO E.coli; low concentration yielded.
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Week 11: 3rd October - 9th October
3rd October | Eloise & Ezekiel
- Set up overnight culture of TaISP in NEB TURBO E.coli for minirpep.
- Transofmred TaISP into NEB T7 Express E.coli.
- Set up overnight culture of Pst_12806 in NEB T7 Express E.coli for protein expression.
4th October | Div, Eloise & Ezekiel
- Miniprep of TaISP in NEB TURBO E.coli.
- Performed day cultures on Pst)12806 NEB T7 Express E.coli for protein expression optimization at different IPTG concentrations and temperatures.
- Harvested the Pst_12806 NEB T7 Express E.coli cultures that were incubated at 30℃ and 37℃ during the day culture; 16℃ culture was incubared overnight.
5th Ocotber | Div, Roger & Ryan
- Harvested the 16℃ culture of Pst_12806 NEB T7 Express E.coli culture from day prior.
- Preformed protein expression check (Bugbuster) for PstSCR1 day culture from the 4th - 5th of October.
- 4 colonies of TaISP in NEB TURBO E.coli were prepared and sent to Ramaciotti Centre of Genomics for sequencing.
6th October | Eloise & Ryan
- Set up overnight culture of TaISP in NEB T7 Express E.coli for protein expression.
7th October | Eloise
- Performed day culture and harvest on TaISP NEB T7 Express E.coli for protein expression.
8th October | Div & Ezekiel
- Performed FRET assay between TaISP and PSt_12806 proteins and MCerulean & mVenus proteins (controls).
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