Team:Heidelberg/Templates/Indigoidine week12

From 2013.igem.org


Contents

Indigoidine production pKH and pMM (Konrad)

IPTG Induction

Induction of 2 days old cultures with/-out 1 mM IPTG. Rosetta seems to produce indigoidine, even without induction (leaky exppression?).

Occulate 2 ml LB + appropiate AB with:

  1. Rosetta + pMM64 + pMM65
  2. BAP1
  3. BAP1 + pMM64
  4. BAP1 + pMM64 + pMM65

Rosetta was oculated from plate (06.07.), the BAP1 culture with 10 µl of ON from 06.07. These DC were incubated for

around 7 hours at 37 °C.

Old broth from 06.07 were induced with 1 mM IPTG at 24 °C for 2 hours. Rosetta strain becomes very blue, even

without IPTG induction.

fusion PCR (bpsA+svp)

based on PCA protocol: Used two main steps, first elongation of fragments (f1;f6) 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 svp as one fragment fused together. The wanted fragment size is ~ 4.6 kbp, so the blue indicated bands were cut out.

Step 1:

  • 13 µl H2O
  • 25 µl Phusion Flash MM
  • 2 µl template (f6:svp)
  • 10 µl template (f1:bpsA from 4.07.)

(concentration of f1 from 4.07. unknown)

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:01; NI:08)
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 0.5 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
  • Gel extraction of ~4.6 kbp bands cut out

Gibson assembly

  • molecular ratio = backbone : inserts = 1 : 3
  • partslength: bpsA=3.8 kbp; svp=0.8 kbp; pSB1C3=2.4 kbp
fragment volume [µl] DNA amount [ng]
f8: bpsA + svp 7.5 214
f7: pSB1C3 (linear.) 2.5 55
total 10
0 µl H2O
10 µl Gibson MM
  • transformation of TOP10 with 2 µl directly of GA reaction mix and 1:4 dilution respectively

additional

  • preparations of JM1 from 4.5 ml TOP10 ON culture (used a bit for transformation, gave rest to Johanna)
  • transformation of BAP1 with 88 ng (5 µl) JM1 (probably way to much DNA)
  • IPTG induction (1mM) experiment with Rosetta + pMM64 + pMM65, BAP1, BAP1 + pMM64, BAP1 + pMM64 + pMM65 (DC, was

in 4 °C fridge overnight)

    • RESLUT: did not turn blue at all, may the IPTG be bad? (did not use the same as the day before)

CPEC Assembly

CPEC for pKH1 assembly: only insert seems visible, no backbone and CPEC product; run at 135 V, 0.8 % gel (TAE); wanted amplicon size is: 2,4 kbp
fragment volume [µl] DNA amount [ng]
f8: bpsA + svp 2.3 150
f7: pSB1C3 (linear.) 5.2 50
total 7.5
2.5 µl H2O
10 µl PhusionFlash MM
total 20
Cycles temperature [°C] Time [min]
1 98 0:10
3 98 0:01
55 0:30
72 2:00
1 72 5:00
1 12 inf
  • use 10 µl for gel picture, store 10 µl

PCR amplification f7 (pSB1C3)

PCR for amplification of pSB1C3: (left) lane 1 & 2: todays amplification, lane 3: Marker, lane 4: 2 µl of gel/nucleotide purified amplification of 4th of July, lane 5 & 6: delRest 11 - 12 with long and short primers (unknown order); (right) gel cut out run at 135 V, 0.8 % gel (TAE); wanted amplicon size is: 2,4 kbp

(add items in this order)

  • 14.6 µl H2O
  • 25 µl Phusion MM
  • 2x 0.5 µl Primer (NI09,NI10)
  • 0.4 µl template (pSB1C3 with J04450 (pJM03))
Cycles temperature [°C] Time [min]
1 98 0:10
30 98 0:01
72 0:40
1 72 5:00
1 12 inf

Ralf

new primers are there yippie

pSB1C3 RB21/22

  • 2,5 ul template pSB1C3 1:5
  • 25 ul Phusion Flash MM
  • 5 ul Primer 10 mM
  • 12,5 ul water
19810
1981
615
7260
29981
7260
172300
112-

T1000 BIORAD

indC-Plum RB27/28

  • 1 ul template cell pellet from DSMZ stem culture
  • 25 ul Phusion Flash MM
  • 5 ul Primer 10 mM
  • 14 ul water
198120
1981
575
7290
29981
655
7290
172300
110-

biometra

sfp-BAP1 RB35/36

  • 1 ul template cell pellet from BAP1 liquid culture
  • 25 ul Phusion Flash MM
  • 5 ul Primer 10 mM
  • 14 ul water
198120
1981
635
7230
29981
655
7230
172300
110-

BioRAD 2-block

Electrophoresis 1 % Agarose 0,5 % TAE 100 V 60 min; PCR product 50 ul + 10 ul 6x loading dye 10 ul quickload 2-log; 2 ul PCR pSB1C3-indC-sfp; 29 ul 2x pSB1C3; 2x indC; 2x sfp

400px|1st run with long primers

400px|cut backbone for gel extraction

indC-Plum #2 Q5 50 ul 1, 2: RB37/38; 3: RB27/28

  • 5 ul template liquid culture - pellet
  • 25 ul Q5 MM
  • 5 ul Primer 10 mM
  • 10 ul water
cyclestemperature °Ctime seconds
198180
30988
5610
72150
172300
112-

sfp BAP1 #2 Q5 50 ul a: RB35/36 b: RB43/44

  • template colony pick
  • 25 ul Q5 MM
  • 5 ul Primer 10 mM
  • 15 ul water
cyclestemperature °Ctime seconds
198180
30988
6210
7240
172300
112-

2-log (1 ug); (1 ul) 1 - 2 - 3 - a - b; (all) 1 - 3 - 3 - a - b - b

2-log (1 ug); (1 ul) 1 - 2 - 3 - a - b; (all) 1 - 3 - 3 - a - b - b

analysis

  • indC fragmet with short primers worked well -> gel extraction and re-PCR with long primers
  • sfp and indC long almost nothing -> improve conditions

sfp BAP1 #3 Q5 50 ul A: RB17/18; naka; 60 °C B: RB17/18; naka; 56 °C C: RB43/44; short; 60 °C D: RB43/44; short; 56 °C E: RB35/36; long; 60 °C F: RB35/36; long; 65 °C

  • template colony pick
  • 25 ul Q5 MM
  • 5 ul Primer 10 mM
  • 15 ul water
cyclestemperature °Ctime seconds
198180
30988
56-60-6510
7230
172300
112-

2-log (1 ug); A-F

analysis

  • B, C and E worked best -> gel extraction
  • re-pcr E

fragments for pRB3

  • rePCR from indC-Plum #2 with long primers
    • 2x 50 ul Phusion Flash HF
    • RB27/28 long primer for pRB3 5 ul 10 uM
    • water 14.8 ul
    • template 0.2 ul pcr gel extracted

biorad T100

cyclestemperature °Ctime seconds
19810
12981
td 57-53 -0.55
7270
18981
655
7270
172300
112-
  • rePCR from sfp-BAP1 #3 E with long primers
    • 2x 50 ul Phusion Flash HF
    • RB35/36 long primer for pRB3 5 ul 10 uM
    • water 14.8 ul
    • template 0.2 ul pcr gel extracted

biorad T100

cyclestemperature °Ctime seconds
19810
30981
655
7220
172300
112-
  • rePCR from pSB1C3 Hanna 1:5 with long primers
    • 2x 50 ul Phusion Flash HF
    • RB21/22 long primer for pRB3 5 ul 10 uM
    • water 14.8 ul
    • template 0.2 ul pcr gel extracted
cyclestemperature °Ctime seconds
19810
12981
td 64 -0.55
7270
23981
655
7270
172300
112-

2-log (1 ug); pSB1C3; indC; sfp

2-log (1 ug); pSB1C3; indC; sfp


assembly pRB3

  • digest 25 ul (2.5 CutSmart 10x; 0.5 enzyme each; 21.5 DNA; 1 h at 37 °C)
    • indC KpnI-HF and BamHI-HF
    • sfp BamHI-HF and NheI-HF
    • pSB1C3 NheI-HF and KpnI-HF
Heidelberg 20130720 RB1-digest.jpg
Heidelberg 20130720 RB1-digest cut.JPG
  • Ligation 1 20 ul (2.0 T4 buffer 10x; 1.0 T4; 10.0 indC; 4.0 sfp; 3.0 pSB1C3)
  • Ligation 2
    • 2.0 T4 buffer 10x; 1.0 T4; 7.0 indC; 10.0
    • 2.0 T4 buffer 10x; 1.0 T4; 10.0 product; 7.0 pSB1C3
Heidelberg 20130720 RB3-ligation.JPG
  • CPEG 20 ul Phusion Flash HF (10 MM; 7 indC; 2 pSB1C3; 1 sfp)

biometra

cyclestemperature °Ctime seconds
19810
10981
3grad. 50 +55
7270
172180
112-
  • Transformation TOP10
    • CPEG 50 5 ul
    • CPEG 55 5 ul
    • CPEG 55 plus 20 min T4 ligase 10 ul (5 ul + 5 ul ligation master mix)
    • CPEG 60 5 ul
    • Lig1 2 ul
    • Lig1 4 ul
    • Lig2 4 ul

TOP10-pRB3-CPEG50-5ul

TOP10-pRB3-CPEG55-5ul

TOP10-pRB3-CPEG55lig-5ul

TOP10-pRB3-CPEG60-5ul

TOP10-pRB3-lig1-2ul

TOP10-pRB3-lig1-4ul

TOP10-pRB3-lig2-4ul

-> order primers for the other PPTases and indigoidine synthetases (set RB21-44 complete)

Results and Discussion

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