Team:CSU Fort Collins/NoteBook/JanThroughApr

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CSU iGEM



January Through April

1/4/16:
  • Ligated b0034 and c0061
  • Transformed Ligated mixture
  • Ran PCR results on a 2% gel
1/5/16:
  • Digested LuxR with EcoRI and SpeI
  • Performed Colony PCR on transformation from the 4th
1/11/16:
  • slr0435 Excision from Synechocystis sp. PCC6803 with PCR using Phusion(size 276 bp).
  • Ran PCR results on a 2% gel
  • Digested I0462 and E0030 with EcoRI and SpeI, R0062 and B0015 with EcoRI and XbaI, B0034 with SPeI and PstI, and C0061 with XbaI and PstI.
1/12/16:
  • Digested slr0435 and resuspended pSB1C3 with EcoRI and PstI
  • Ligated pSB1C3 and slr0435
  • Transformed ligation mixture
1/13/16:
  • Colony PCR performed on slr0435+pSB1C3 transformed colonies
  • Ran PCR results on 1% gel
  • Digested I0462 and E0030 with EcoRI and SpeI, R0062 and B0015 with EcoRI and XbaI, B0034 with SPeI and PstI, and C0061 with XbaI and PstI.
  • Ran on 1% gel
1/21/16:
  • Digested I0462 and E0030 with EcoRI and SpeI, and C0061 with XbaI and PstI.
  • Ran on 1% gel
  • Phosphatase treatment
  • Use Antarctic Phosphatase with 10x buffer. Need 1uL to treat 1ug of DNA
  • Add 1uL phosphatase and 5uL 10x buffer to bring total rxn volume to 50uL
  • Incubate at 37C for 1 hour, and heat inactivate at 70C for 5 minutes
1/25/16:
  • Overnight ligated R0062 and I0462, B0034 and C0061, B0015 and E0030 at 16 C
1/25/16:
  • Transformed the overnight ligation mixtures
2/6/16:
  • PCRed out BBA_J23106 out of glycerol stock 19 using Q5 master mix 19rxns
2/9/16:
  • Transformed Bba_K325909 from parts kit into DH5a cels
2/11/16:
  • To confirm that Bba_K325909 are functioning we tried to induce expression using L-arabinose
  • No luminescence was produced
2/16/16:
  • Decided to order two gBlocks for quorum portion of the project
3/24/16:
  • Set up O/N cultures of K1033929, K592009 and E1010 in 5 mL of LB with 5 uL of Cm34 antibiotic
3/29/16:
  • Ran promoters Sll027, and LrtA in a 1% gel
    • Sll0027
    LrtA
4/6/16:
  • Ran Cpcg2 on 1% gel
4/12/16:
Prepare pSB1C3 vector for Infusion
  • gBlocks (Quorum 1 and Quorum 2) have 15 bp homology with each other along with prefix and suffix at the entire block’s ends
  • Digesting the vector with XbaI will leave 16 bp homology with Quorum 1
  • Digesting the vector with SpeI will leave 20 bp homology with Quorum 2
  • Digested c0061 with SPeI and XbaI
  • Ran on 1% gel
4/12/16:
  • Reaction: PSB1C3 3.06 uL,Q1 2.5ul,Q2 1.78ul, 5x Infusion Mix 2ul, H20 .66ul
    • Transformation
  • Add 1 uL Infusion rxn to 50 uL Stellar Cells
  • Incubate 30 min on ice, 45 sec at 42C, 2 min on ice
  • Add 250 uL SOC media
  • 1 hour outgrowth at 37C
  • Plate 50 uL on LB+Cam plates
  • 37C overnight
4/18/16:
  • Colony PCR on 5 Qblock transformed colonies
4/19/16:
  • Ran PCR products from the 18th on a 1% gel
4/20/16:
  • Quorum Colony PCR Phusion
4/22/16:
  • Ran the PCR product from the 20th on a 1% gel

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