Team:Heidelberg/Templates/Indigoidine week12

From 2013.igem.org

Latest revision as of 00:46, 5 October 2013

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)

• 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

• 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

• 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))

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

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

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

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

cycles	temperature °C	time seconds
1	98	180
30	98	8
	56	10
	72	150
1	72	300
1	12	-

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

cycles	temperature °C	time seconds
1	98	180
30	98	8
	62	10
	72	40
1	72	300
1	12	-

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

cycles	temperature °C	time seconds
1	98	180
30	98	8
	56-60-65	10
	72	30
1	72	300
1	12	-

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

cycles	temperature °C	time seconds
1	98	10
12	98	1
	td 57-53 -0.5	5
	72	70
18	98	1
	65	5
	72	70
1	72	300
1	12	-

• 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

cycles	temperature °C	time seconds
1	98	10
30	98	1
	65	5
	72	20
1	72	300
1	12	-

• 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

cycles	temperature °C	time seconds
1	98	10
12	98	1
	td 64 -0.5	5
	72	70
23	98	1
	65	5
	72	70
1	72	300
1	12	-

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

• 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

• CPEG 20 ul Phusion Flash HF (10 MM; 7 indC; 2 pSB1C3; 1 sfp)

biometra

cycles	temperature °C	time seconds
1	98	10
10	98	1
	3grad. 50 +5	5
	72	70
1	72	180
1	12	-

• 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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