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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

• Gibson results: plate agar was dry. Colony PCR will be done of surviving colonies.

• PCR Run

Name PCR Template Primer F Primer R Length 1 pBAD-ADF-3 ADF-3 16.F9 F8 2000 2 pBAD-ADF-3 ADF-3 16.F9 F8 2000 3 ADF-3+ VB LuxBrick 16.F7 F10 3000 4 ADF-3+ VB LuxBrick 16.Fn F10 3000 5 tap-BB Cyano DNA 16.I1 I4 140-190 6 LuxCDEG (sigEP) K3259015 16.H1 H4 4000 7 LuxCDEG (sigEP) K3259005 16.H1 H4 8 LuxCDEG (pcaa3) K3259015 16.H9 H4 4000 9 LuxCDEG (pcaa3) K3259005 16.H9 H4 10 pcaa3 LuxCDEG - VB IntC psbA2-IntC 16.H3 16.H8 4000 11 psigEP LuxCDEG – VB IntC psbA2-IntC 16.H3 16.G10 4000 12 LuxAB K328905 16.G5 16.G8 2000 13 LuxAB K352905 16.G5 16.G8 2000 14 LuxAB - VB ->Need C K

Notes

• Bands 6 and 8 were really faint but had the right size.
• Amplicons in bold

Friday

• Band purification

Name PCR Concentration [ng/µl]

1 pBAD-ADF-3 9.7 6 LuxCDEG (sigEP) 5.6 8 LuxCDEG (pcaa3) 4.1 10 pcaa3 LuxCDEG - VB IntC 14.8 11 psigEP LuxCDEG – VB IntC 33.2 13 LuxAB (Xenor..lumins..) 6.1

Notes Parts 6 and 8 (LuxCDEG) will be used for a new PCR because they were very faint.

• Gibson Isothermal Buffer

A new buffer was made because we realized the old one was prepared with wrong concentrations. New concentrations: MgCL2 2M, DTT 1M.

• Gibson and transformation

C1.1: C4-Open+pSBAB+mRFP (CK) C1.2: C4-Open+pSBA2+mRFP (CK) Pcaa3 BB: Pcaa3 BB + VB_Pcaa3 (C) ADF 3: ADF3 + (pBAD) VB_ADF3 (C)

• Error: mRFP was used for C1.2 instead of RFP (C1.2 is designed to work with RFP).
• C4 Open: RS1+KAN+B0015+RS2

• Colony PCR

C1.1 and C1.2 (assembly date 08.01): 20 colonies were selected from each plate. Right sized colonies: 5,6,7,8 and eleven from C1.1 and 11 from C1.2. Maybe colony 27 from C1.2. This Gibson assembly was repeated anyway. Colony 6 did not grow.