Team:SPSingapore/Notebook-Week-8

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  • Week 1 (24/5 - 30/5)
  • Week 2 (31/5 - 6/6)
  • Week 3 (7/6 - 13/6)
  • Week 4 (14/6 - 20/6)
  • Week 5 (21/6 - 27/6)
  • Week 6 (28/6 - 4/7)
  • Week 7 (5/7 - 11/7)
  • Week 8 (12/7 - 18/7)
  • Week 9 (19/7 - 25/7)
  • Week 10 (26/7 - 1/8)
  • Week 11 (2/8 - 8/8)
  • Week 12 (9/8 - 15/8)
  • Week 13 (16/8 - 22/8)
  • Week 14 (23/8 - 29/8)
  • Week 15 (30/8 - 5/9)
  • Week 16 (6/9 - 12/9)
  • Week 17 (13/8 - 17/9)

Research Notebook

Week 8 (12/7 - 18/7) ▪ July 13 Anaerobic Promoter    ☀ Yi Han & Yun Ting ☀ Ligation for FNRgfp (4X) 200ng of gfp plasmid (4.36ul) 297.5 ng insert (7:1) 10.3 1ul 10Buffer (53.34) 4ul T4 ligase Trial run of anaerobe chamber open sachet to decrease O2 at 3.30pm takes 2.5h to activate streak plate of P. putida a strict aerobe and E. coli, facultative aerobe in chamber at 30deg C to grow O/N P. putida is an obligate aerobe and if chamber works ,it will not grow E. coli should grow in both conditions controls grown outside chamber ▪ July 14 Anaerobic Promoter    Invasin + Listerolysin ☀ Yi Han ☀ No colonies for FNRgfp plasmid Inv Plasmid Verification buffer 1 EcoRI 0.5 in plasmid 0.5 dH2O 8 Total 10ul Gel extraction RE digest of EsaR-GFP plasmid Ligation Control Vector (45ng) 1 1 insert 1 1 Buffer 1 1 dH2O 6 7 ligase 1 1 Transformation of FNRgfp Anaerobe jar E. coli grew in both conditions P putida-> some growth in anaerobe chamber Proper streak plate in aerobic conditions Packet runs out after 16 hours ▪ July 15 Anaerobic Promoter    ☀ Yi Han ☀ FNRgfp-> no colonies after transformation religation (4X reaction) 200ng of gfp plasmid (4.4ul) 2125ng of insert (8ul) 4ul T4 ligase 55.6 H2O Anaerobic Promoter    ☀ Yi Han & Chi Yan ☀ Transformation of FNR gfp into 20ul of BL21 ▪ July 16 Anaerobic Promoter    ☀ Yi Han ☀ No colonies grew Ran a gel, FNRgfp pcr, gfp pcr, fnr, 100bp ladder ▶ Seems to be band shift. ▪ July 17 Anaerobic Promoter    ☀ Chi Yan ☀ RE digest of FNR-GFP fusion PCR with EcoRI and PstI EcoRI 1 PstI 1 Buffer 5 FNR-GFP from 12/7 in RIP box 1ug -> 2.5ul H2O 40.5 Total 50 ▶ RE for 2 hours at 37 degrees ▶ Direct purification using Promega kit Overnight ligation (4X reaction) 200ng of gfp plasmid (4.4ul) 400ng of insert (5:1) (4ul) 4ul T4 ligase 8ul T4 ligase buffer 44.1 H2O ESA Quorum Sensing    ☀ Adrian ☀ Ligation of esaRBS-GFP (EcoRI/PstI) and RNG-GFP (EcoRI/PstI) into pSB1A2 (EcoRI/PstI) 10x T4 DNA ligase Buffer 1ul Vector 1ul Insert 5ul H2O 10ul T4 DNA ligase 1ul ▶ Reactions were incubated for 30min at room temperature Transformation   pSB1A2 esaRBSGFP   pSB1A2 RNG GFP   Control cut pSB1A2