Team:Heidelberg/Templates/Indigoidine week13

From 2013.igem.org

Indigoidine production Konrad (Konrad)

PCR for amplification of pSB1C3: (left) first analytical gel; (right) second analytical gel. f7 was twice eluted after gel extraction, the second run was 30 min in 15 µl H₂O at 50 °Ca before centrifugation; run at 100 V, 0.8 % gel (TAE);

• gel extraction of yesterdays fragment f7:pSB1C3
• analytical gel (small, half size): (M: 3 µl NEB log2, 2 µl f7 + 2 µl 1x loading dye)
  ==> [f7] = 3 ng/µl
• 2nd analytical gel (small, half size): (M: 3 µl NEB log2, (2 µl f1:4.07., 2 µl f7:14.07., 2 µl f7:14.07.-2nd

eluation, 2 µl delRest-f6-9) + 8 µl 1x loading dye)
==> [f1:4.07.] = 18 ng/µl, [f7:14.07.] = 5 ng/µl,

[f7:14.07.-2nd] = 2 ng/µl,[delRest-f] = 5 ng/µl

fusion PCR (bpsA+svp)

based on PCA protocol: Used two main steps, first elongation of fragments (f1;f7) without any primers, than

enrichment of desired fusion product with fw primer of fragment 1 and rv primer of fragment 2.

PCA in order to gain bpsA and pSB1C3 as one fragment fused together. The wanted fragment size is ~ 6.2 kbp.

Step 1:

• 10 µl Phusion Flash MM
• 6.3 µl template (f7:pSB1C3)
• 3.7 µl template (f1:bpsA from 4.07.)

Step 2:

• 20.0 µl H₂O
• 25 µl Phusion Flash MM
• 4.0 µl template (reaction from step 1)
• 2x 0.5 µl primer (NI:06; NI:09)

Preparation for electroporation

analytical gel of purified Gibson assembly and delRest;run at 100 V, 0.8 % gel (TAE);

• Gibsonassembly purified with Nucleotide Removal Kit
• analytical gel (small, half size): (M: 2.5 µl NEB log2)
  ==> [delRest(short)] = 6-7 ng/µl, [delRest(long)] = 4

ng/µl, [GA] = 3 ng/µl

Validation of Gibson assembly

Gibson assembly. The wanted fragment size is ~ 7.0 kbp.;run at 100 V, 0.8  % gel (TAE);

• put 10 µl on gel.
• electroporation with left 4 µl

fusion PCR (bpsA+svp)

PCA in order to gain bpsA and pSB1C3 as one fragment fused together. The wanted fragment size is ~ 6.2 kbp.;run at 135 V, 0.8 % gel (TAE);

Redo 2nd step with 1st step result of the 13th of July

Step 2:

• 23.5 µl H₂O
• 25 µl Phusion Flash MM
• 0.5 µl template (reaction from step 1)
• 2x 0.5 µl primer (NI:06; NI:09)

soil sample preparation

• experimental CPEC assembly with left mix from day before

Analytical gel for verification of CPE success.

File:Heidelberg_20130719-CPEC.png

• PCR of bpsA and svp with new Primers

PCR amplification of svp with new Primers.

PCR amplification of bpsA with new Primers.

• gel extraction of new bpsA and svp fragments

Analytical gel bpsA and svp gel extraction.

• Gibson Assembly
• CPEC

Analytical gel for verification of Gibson assembly and CPEC success.

• Trafo

• Colony PCR of electroporation with GA

Colony PCR for pKH1 validation. The T-TE domain of bpsA was used as amplicon (size: 1.1 kbp) as well as svp-part of backbone (similar size of 1.1 kbp). Every lane represents one PCR reaction with 1 picked colony each. Except for colony 1 only black colonies were picked.

• Colony PCR of trafos of day before

Colony PCR for pKH1 und pKH2 validation. The T domain of bpsA was used as amplicon (size: 300 bp). Every lane represents one PCR reaction with 3 picked colony each.

• Heat-shock competent Rosetta
• Minipreparation of 2 ml ON culture of one picked black colony of GA plate (electroporation)

Ralf

PCR PPTases #1 Phusion Flash HF 50 ul (long) 20 ul (short); colony pick/0.2 ul pcr template; primer 2/5 ul 10 uM

• MG1655 pick RB33/34 and RB 41/42
• S. vert pick liquid RB29/30 and RB 39/40
• pMM65 pcr RB25/26

biorad T100

PCR bpsA64 Phusion Flash HF 50 ul (long); 0.2 ul pcr template; primer 5 ul 10 uM

• pMM64 Fussenegger miniprep RB23/24

biometra

bpsA; entD-l; svpV-l; svpF-l; entD-s; svpV-s

bpsA; entD-l; svpV-l; svpF-l; entD-s; svpV-s

PCR PPTases #3 Phusion Flash HF 50 ul (25 MM; 5/5 primer; 14.5 water; 0.5 template)

• 1: entD from PPTases #1 gel ex w/ RB33/34 long
• 2: svpV from ??? w/ RB29/30 long
• 3: svpF from PPTases #1 gel ex w/ RB25/26 long

1 ug 2-log; entDlong; svpVlong; svpFlong

1 ug 2-log; entDlong; svpVlong; svpFlong

analysis

• entD and svpF -> gel extraction
• svpV is too small -> wrong product. new colony PCR with short primers

Konrad PCR svpV #3

2-log; RB13/14 65°C; RB39/40 62 °C; RB 39/40 57 °C

analysis

• hmm

PCR svpV #4 20 ul Phusion Flash HF (10 MM; 9.4 water; 0.2 ul primer 100 uM; 0.2 cell pellet) conditions as in Jul 12th; RB13/14 biorad T100

2-log; Hanna; svpV RB13/14

2-log; Hanna; svpV RB13/14

rePCR svpV #5 from #4 50 ul (25 MM; 14 water; 0.5 primer 100 uM; 0.2 template)

• 1: RB29/30 from short RB13/14
• 2: RB29/30 from short RB 39/40

biorad T100

2-log; from taka; from RB39/40

2-log; from taka; from RB39/40

-> gel extraction; CPEG assembly

CPEG assembly 20 ul Phusion Flash HF (10 MM; 2 pSB1C3; 1.5 PPTase; 6 ind synthetase) biometra

Transformation 10 pm; 80 ul +/- 20 ul; 5 ul CPEG pcr product

• medium LB: FG 2013Jul19
• plates LB+Cm: FG 2013Jul22
• TOP10 from stock -80 °C

Results and Discussion