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From 2012.igem.org

Monday:

• Band purification of LuxCD & Lux EG.

• Gel electrophoresis: Colony PCR of RS2 cut (no extra restriction site) and Luc CDEG. Right sized bands: 11 and 12. Plasmids from preceding colonies were extracted and sized was checked by electrophoresis. Finally they were ligated to B0014 and B0015.

Tuesday:

• PCR Run

VB psb1C3, VBpsb1A3, VB psb1T3, VB psb1K3, V psb1K5, Lux CD

• Colony PCR

RS1+RS2: (12 colonies selected) B0014+RS2: (12 colonies selected) B0015+RS2: (12 colonies selected) Lux CD+Lux EG: (4 colonies selected) Positive and negative control

Results: correct controls. Most of the colonies were right sized. Error in Lux CD+EG because the bands were much smaller than expected (1000 bp). Two colonies were chosen in each case. Digestions left overnight: Lux CD from Brick, and Lux EG obtained by PCR. Wrong digestions were eliminated.

Wednesday:

Overnight digestions were finished and 20 μL were run in gel (1%). Digestions showed right size and high DNA concentration. Part Lux CDEG was ligated, transformed and left growing.

• Colony PCR purification of slected colonies

RS1+RS2 (colonies 2 and 6) B0014+RS2 (colonies 2 and 6) B0015+RS2 (colonies 20 and 23)

Desired parts were obtained.

• Digestions

RS1+RS2 (E+P) B0014+RS2 (X+P) B0015+RS2 (X+P)

With the last two and RS1+Kan (E+s). The following parts were ligated and transformed:

RS1+Kan+B0014+RS2 in pSB1C3 RS1+Kan+B0015+RS2 in pSB1C3

These were used to insert parts between RS1 and Kan through Gibson Assembly.

• Amplication and purification of plasmids: PSB1C3, pSB1T3, pSB1K3, pSB1A3, pSB1K. They were very faint and their DNA concentration was vwery low, therefore the plasmids were dismissed. The only one that was kept was pSB1K5 (24ng/μl)

• PCR run:

Name PCR Template Primer F Primer R

B1C VB-pBAD LuxBrick 16.F7 16.F10 B2C VB-pBAD LuxBrick 16.F7 16.G2

B1	ADF3	          ADF3	                16.F9	        16.F8	   
B2	ADF3	          ADF3	                16.G1	        16.F8	   

C1 RS1+RS2 RS1+RS2 14.F2 14.G2

C1A	pSB1A3 for gibson Linearized VB pSB1A3	14.F3	        14.G1	   
C1T	pSB1T3 for gibson Linearized VB pSB1T3	14.F3	        14.G1	   

LCD Lux CD Lux CD VF2 VR pSB1A3 pSB1A3 Linearized VB pSB1A3 Suffix F Preffix R

Notes B1C, B2C, C1, LCD and pSB1A3 were right sized and were miprepped afterwards (elution in 20 μL). Only B1C (VB-pBAD) and RS1+RS2 yielded high concentrations (25,9 ng/μL and 73,1 ng/μL) respectivamente. A new PCR will be done to amplify mipreps with low concentrations.

Thursday:

• Colony PCR for colonies with LuxCDEG in pSB1K3 and RS1+Kan+B0014/B0015+RS2 in pSB1C3. They turned out to be the right size. Colonies 9 and 11 were selected from RS1+Kan+B0015+RS2 and colony 14 for RS1+Kan+B0014+RS2. Both colonies were slected for CDEG although the size is not confirmed to be the adequate. After miniprep the correct ligation will be tested.

• PCR Run

Name PCR Template Primer F Primer R

B1 ADF-3 ADF-3 16.F9 16.F8 B2 ADF-3 ADF-3 16.G1 16.F8 C1A VB(pSB1A3) for gibson Linearized VB pSB1A3 14.F3 14.G1 C1T VB(pSB1T3) for gibson Linearized VB pSB1T3 14.F3 14.G1 A3 pSB1A3 Linearized VB pSB1A3 Suffix F digestion Preffix R digest T3 pSB1T3 Linearized VB pSB1T3 Suffix F digestion Preffix R digest C3 psb1C3 Linearized VB psb1C3 Suffix F Prefix R LAB LuxAB Lux AB 15.G2 15.G5

Gel will be run tomorrow. pSB1C3 will be included as it was forgotten today.

Friday

• Minipreps and concentrations

Lux CD --> 16,6 ng/μl Lux CDEG (from 1) --> 64,0 ng/μl RS1+KanR+B0015+RS2(from 9) --> 83,6 ng/μl RS1+KanR+B0015+RS2(from 11) --> 60,7 ng/μl RS1+KanR+B0014+RS2 (from 14) --> 42,6 ng/μl

• Band purification

psb1C3 --> 6,3 ng/μl ADF3 --> 10,1 ng/μl

Digetsion verifications were done with E + P and then gel electrophoresis

(1) has wrong size (probably due to: it was just lux CD) (9) has wrong size (11) right size. Construct C4 is correctly assembled. Sequence will be done to confirm. (14) has wrong size.

Further plans:

Basically we have three problems:

1. We are not sure what primers amplified the parts. If they were done with preffix and suffix the restriction enzymes will not cut our parts.

2. When parts CD and EG are cut, many pieces are left around and as the ligase also ligates blunt ends many false positives are generated.

3. CD, EG and psb1C3 have the same size. We cannoyt separate by electrophoresis.

So, we developed to ways to assembly Lux CDEG:

Way 1

1. amplify CD and EG with VF2 and VR 2. Digest: - Lux CD (E+P),LuxEG (X+P), pSB4K5 (E+P) 3. Electrophoresis (parts can be distinguished now) 4. Ligate three parts.

Way 2

1. Digest LuxCD from plasmid (S+P) 2- Ligate with Lux EG obtained by Way 1

Several PCR's were left overnight: transaldolase promoter, Pcaa3, Lux AB and several VB (psb1C3,psb1K3,psb1A2,psb1T3).