• Ligated b0034 and c0061
• Transformed Ligated mixture
• Ran PCR results on a 2% gel

• Digested LuxR with EcoRI and SpeI
• Performed Colony PCR on transformation from the 4th

• 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.

• Digested slr0435 and resuspended pSB1C3 with EcoRI and PstI
• Ligated pSB1C3 and slr0435
• Transformed ligation mixture

• 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

• 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

• Overnight ligated R0062 and I0462, B0034 and C0061, B0015 and E0030 at 16 C

• Transformed the overnight ligation mixtures

• PCRed out BBA_J23106 out of glycerol stock 19 using Q5 master mix 19rxns

• Transformed Bba_K325909 from parts kit into DH5a cels

• To confirm that Bba_K325909 are functioning we tried to induce expression using L-arabinose
• No luminescence was produced

• Decided to order two gBlocks for quorum portion of the project

• Set up O/N cultures of K1033929, K592009 and E1010 in 5 mL of LB with 5 uL of Cm34 antibiotic

• Ran promoters Sll027, and LrtA in a 1% gel

Sll0027



LrtA

• Ran Cpcg2 on 1% gel

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

• 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

• Colony PCR on 5 Qblock transformed colonies

• Ran PCR products from the 18th on a 1% gel

• Quorum Colony PCR Phusion

• Ran the PCR product from the 20th on a 1% gel