Team:Heidelberg/Templates/Indigoidine week13

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Revision as of 00:32, 5 October 2013


Contents

Indigoidine production Konrad (Konrad)

File:20130714-f7-delRest.png
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 °C 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.

File:090714-f9.png
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)
Step Cycles temperature [°C] Time [min]
1 1 98 0:10
5 98 0:01
52 0:30
72 0:20
1 72 5:00
1 12 inf
use only 4.0 µl of mixture for 2nd step
2 1 98 0:10
30 98 0:01
72 1:10
1 72 5:00
1 12 inf

Preparation for electroporation

File:20130714-GA.png
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

File:20130715-GA.png
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)

File:20130715-PCA.png
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)
Step Cycles temperature [°C] Time [min]
2 1 98 0:10
30 98 0:01
72 1:10
1 72 5:00
1 12 inf

soil sample preparation

  • experimental CPEC assembly with left mix from day before
File:20130719-CPEC.png
Analytical gel for verification of CPE success.

File:20130719-CPEC.png

  • PCR of bpsA and svp with new Primers
File:20130719-svp new.png
PCR amplification of svp with new Primers.
File:20130719-bpsA new.png
PCR amplification of bpsA with new Primers.
  • gel extraction of new bpsA and svp fragments
File:20130719-GE-f8 10 11-delRest.png
Analytical gel bpsA and svp gel extraction.
  • Gibson Assembly
  • CPEC
File:20130719-GA-CPEC.png
Analytical gel for verification of Gibson assembly and CPEC success.
  • Trafo
  • Colony PCR of electroporation with GA
File:20130720-CP GA electroporation.png
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
File:20130721-delRest4-7 21-9 colony.png
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

cyclestemperature °Ctime seconds
198180/10
12981
td 68 -0.55
7220
18981
655
7220
172180
112-


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

  • pMM64 Fussenegger miniprep RB23/24

biometra

cyclestemperature °Ctime seconds
19810
12981
td 58 -0.55
7270
18981
655
7270
172300
112-
20130724 pMM64-RB-3set.jpg

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

20130724 pMM64-RB-3set cut.jpg

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
cyclestemperature °Ctime seconds
19810
30981
655
7220
172180
112-
20130726 PPTasenhasen.jpg

1 ug 2-log; entDlong; svpVlong; svpFlong

20130726 PPThasen cut.jpg

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

cyclestemperature °Ctime seconds
198180
30981
3grad. 65-62-575
7220
172180
110-
20130726 svpV.jpg

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

cyclestemperature °Ctime seconds
198120
30981
655
7220
172300
112-
20130728rb svpverttaka.jpg

2-log; Hanna; svpV RB13/14

20130728rb svpverttaka cut.jpg

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

cyclestemperature °Ctime seconds
19810
10981
td 65 -0.55
7220
25981
655
7220
172300
112-
20130728 resvpVlong.jpg

2-log; from taka; from RB39/40

20130728 resvpVlong cut.jpg

2-log; from taka; from RB39/40

-> gel extraction; CPEG assembly

plasmid namebackbonecutting siteindigoidine-Synthetasecutting sitePPTasecutting siteprimers
pRB3pSB1C3-BBa_B0034KpnIP.lum indCBamHIBsub sfpNheIRB21/22-27/28-35/36
pRB4pSB1C3-BBa_B0034KpnIP.lum indCBamHISvert svpNheIRB21/22-27/28-29/30
pRB5pSB1C3-BBa_B0034KpnIP.lum indCBamHISvert svp65NheIRB21/22-27/28-25/26
pRB6pSB1C3-BBa_B0034KpnIP.lum indCBamHIEco entDNheIRB21/22-27/28-33/34
pRB7pSB1C3-BBa_B0034KpnIS.lav bpsA64BamHIBsub sfpNheIRB21/22-23/24-35/36
pRB8pSB1C3-BBa_B0034KpnIS.lav bpsA64BamHISvert svpNheIRB21/22-23/24-29/30
pRB9pSB1C3-BBa_B0034KpnIS.lav bpsA64BamHISvert svp65NheIRB21/22-23/24-25/26
pRB10pSB1C3-BBa_B0034KpnIS.lav bpsA64BamHIEco entDNheIRB21/22-23/24-33/34


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

cyclestemperature °Ctime seconds
19810
10981
555
7270
172180
112-

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

Retrieved from "http://2013.igem.org/Team:Heidelberg/Templates/Indigoidine_week13"