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From 2012.igem.org

• Batch full of competent cells. OD: 0.495

• Ligation

LuxCD +LuxEG in pSB4K5 (L1) and pSB1K3 (L2).

• Gibson: sfGFP in pSB4K5. Assembly made to test new competent cells

• Transformation

Plates

L1: Ligated LuxCD +LuxEG in pSB4K5 at 1:1 and 1:10 L2: Ligated LuxCD +LuxEG in pSB1K3 at 1:1 and 1:10 G+: Gibson sfGFP in pSB4K5 PUC19 (AMP) x3: to test our new competent cells, 20µL of transformed bacteria were plated.

Tuesday

• Transformation results

PUC19-1: 148 colonies PUC19-2: 177 colonies PUC19-3: 155 colonies L1 and L2 with colonies. Colony PCR will be done. G+ Positive results. Several colonies. Probably due to: the problem with the Gibson assembly could be the use of E. cloni instead of TOP10.

• Colony PCR for LuxCDEG in pSB1K3: Negative. PCR will be tried.
• Colony PCR for LuxCDEG in pSB4K5 was not done due to all colonies that grew were red. It was assumed LUXCDEG did not integrate.
• PCR for RS1-Kan-B0015-RS2 (chloramphenicol resistance) and electrophoresis: primers form dimers. PCR will be repeated at higher Tm.

• PCR run:

Name PCR Template Primer F Primer R Length

1 RS1 KAN B0015 RS2 F8 F5 4300

2 KAN B0015 RS2 F8 F2 3760

Notes

• It was a touch-Down PCR

1 and 2 amplified and had right size. Only 1 will be purified because is more useful than 2.

Wednesday

• Band purification

Name PCR date Concentration ng/µl

C4-Open 07.31 21,2 Pcaa3 promoter 07.27 18,1

• PCR run:

Name PCR Template Primer F Primer R Length 1 SigEP-LuxCDEG K325005 16.H1 16.H4 4000 2 Pcaa3-LuxCDEG K325005 16.H9 16.H4 4000 3 SigEP 4 LuxCDEG Cyano 16.G9 16.H2 4 Pcaa3 4 LuxCDEG Cyano 16.H10 16.H7 101-141 5 taP (BB) Cyano 16.I1 16.I4 150-190 6 taP_VB pSB1C3 16.I2 16.I3 2000 7 Pcaa3 (BB) Cyano 16.I5 16.I8 101-141 8 Pcaa3_VB pSB1C3 16.I6 16.I7 2000 9 taP-LuxAB Cyano 16.G3 16.G6 150-190 10 LuxAB in CK K325009 16.G5 16.G8

• Gibson assembly:

C1.1: C4-Open+pSBAB+mRFP (CK) C1.2: C4-Open+pSBA2+mRFP (CK)

Thursday

• Band purification

Name PCR Concentration [ng/µl]

1 SigEP-LuxCDEG 26.1 2 Pcaa3 4LuxCDEG 8.8 3 taP_VB 12.6 4 Pcaa3_VB 20.3 5 taP-LuxAB 26.6 6 ADF-3+ 13.0 7 Pcaa3 4 BB 13.8

Notes: amplicons in bold