Indigoidine production with pKH1 II (Konrad)

fragment amplification

==> f1: bpsA (all)

• 14.6 µl H2O
• 25 µl Phusion Flash
• 2x 0.5 µl Primer (NI01,NI06)
• 0.4 µl template pMM64

Gel purification (50µl): by accident did not take 3 but 1 Volume buffer QG

Labelling fragements: 1 I and 1 II (from different lanes)

fusion PCR

==> for fusion of both fragments bpsA and svp using Phusion Flash HF

• 19.8 µl H2O
• 25 µl Phusion Flash MM
• 2x 1.0 µl Primer (NI01,NI08)
• 2.6 µl template (f1:bpsA)
• 0.6 µl template as 1:10 dilution (f6:svp)

Gel purification (50µl): by accident did not take 3 but 1 Volume buffer QG

Labelling fragments: 1 I and 1 II (from different lanes)

Amplification pSB1C3 (f7)

==> f7: pSB1C3 (linear.) with our Phusion MM and Phusion MM of SYNtheSYS (old, don't know if functional...)

(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))

-->Hot start

RESULT: no product neither with Synthesys fusion nor our fusion

==> f7: pSB1C3 (linear.) with Fusion Flash

(add items in this order)

• 14.6 µl H2O
• 25 µl Phusion Flash
• 2x 0.5 µl Primer (NI09,NI10)
• 0.4 µl template (pSB1C3 with J04450 (pJM03))

fusion PCR

==> for fusion of both fragments bpsA and svp using Phusion Flash HF

• 19.8 µl H2O
• 25 µl Phusion Flash MM
• 2x 1.0 µl Primer (NI09,NI10)
• 5 µl template (f1:bpsA I,03.07.13)
• 0.6 µl template as 1:10 dilution (f6:svp)

--> wrong primers, should have taken NI01 and NI08
--> more than 50µl as not enough bpsA template was left

Re-PCR of fusion (bpsA+svp)

==> for amplification of fusion fusion product 03.07 (bpsA + svp) using Phusion Flash HF

• 18 µl H2O
• 25 µl Phusion Flash MM
• 2x 1.0 µl Primer (NI09,NI10)
• 5 µl template (fusion bpsA+svp,03.07.13)

--> wrong primers, should have taken NI01 and NI08

Re-PCR of fusion (bpsA+svp)

==> for amplification of fusion fusion product 03.07 (bpsA + svp) using Phusion Flash HF

• 19 µl H2O
• 25 µl Phusion Flash MM
• 2x 0.5 µl Primer (NI01,NI08)
• 5 µl template (fusion bpsA+svp,03.07.13)

Preparations

• prepare liquid culture of Rosetta + pMM64 + pMM65 in 2 ml LB with Amp + Kan for plate oculation
• prepare liquid cultures of TOP10 + pMM64/65 for minipreps: 3 ml TB + Amp/Kan
• prepare liquid cultures for induction test: 3 ml LB + appropiate AB
  • BAP1
  • BAP1 + pMM64 (Amp)
  • BAP1 + pMM64 + pMM65 (Amp + Kan)
  • TOP10 + pJH1 (Kan)

Re-PCR of fusion (bpsA+svp)

Ralf

PCR from glycerol stock S. lavendulae with Fermentas; Thermoscientific and NEB Taq 50 ul

• MM 25 ul, 7,5 ul primer RB7/8 100 mM, 5 ul water, 5 ul culture

• S. verticillus and S lavendulae in 50 ml YEME medium for 46 h at 28 °C.
• filamentous growth and spherical colonies in S. lavendulae flask after 1 day -> contamination?!
• no growth in S. verticillus flask after 18 h
• prepare plates

colony PCRs

colony PCRs 50 ul Phusion flash (25 ul + 5 ul Primer 10 mM + 10 ul water

• bpsA - S. lav (Takahashi) RB11/12
• svp - S. vert (Taka Primer) RB13/14
• svp - S. vert (Sanchez) RB15/16
• entD - MG1655 (Lambalot) RB19/20
• sfp - BAP1 RB17/18

biometra

Gel

Gradient PCR with both Streptomyces under same conditions to validate PCR parameters. Phusion Flash HF 20 ul (10 ul MM; 2 ul Primer 10 mM; 2 uL template (cell pellet from GYM 48 h liquid culture; 6 ul

H2O)

Gel

===Results and Discussion===

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===

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===

===Konrad===

validation of ccdB constructs

• MP of pKH2(ccdB) constructs from independent colonies: 3rd and 4th were used according to colony PCR result
  • (elution in 30 µl): col3=170 ng/µl ; col4=145 ng/µl
• testtrafo in ccdB resistent Survival, TOP10 and DH5alpha cells (from Dominik) of constructs pKH2, pKH2(ccdB)-

col3, pKH2(ccdB)-col4

• • transformation with 20 ng
  • plated 30 µl of cell suspension on 3 partiated plates for each constructs

Construction of ccdb-constructs pRB11-13

pRB11, pRB12 (bpsA+svpF) and pRB13 (indC+sfp) carry the ccdb gene with RBS instead of their native T-Domain so that

they don't produce the blue pigment, survive in OneShot ccdb survival cells and die when transformed to TOP10 or

DH5alpha. If the new T-Domain is successfully inserted, transformed cells survive in TOP10 and hopefully produce

the blue pigment.

amplification of CPEC fragments

NI primer in this case are for pRB11 and pRB12; KH primer for pRB13 templates are miniprepped plasmids

• ccdb (NI13/14; KH1/2; 350 bp)
• construct pKH1/2 and pRB3 without T-Domain (NI15/16; KH3/4; 7000 bp)
• indC T-Domain as a control (NI17/18; KH5/6; 200 bp)
• bpsA T-Domain as a control (NI19/20; KH7/8; 200 bp)

Agarose Gel and Gel Extraction

For NI15/16 wrong template (pMM64) was used in the first PCR. The second run (pKH1/2) yielded the right product fragments have been gel extracted using QIAquick Gel extraction kit. NI15/16 and KH3/4 have been extracted using

QiaEXII gel extraction kit.

CPEC assembly and Transformation

CPEC assembly of CCDB fragment (KH1/2 and NI13/14) and indigoidine synthesis construct (pRB3-delta-T, pKH1-delta-T

and pKH2-delta-T to yield pRB11, pRB12 and pRB13. As a positive control, native T-Domains (KH5/6 and NI19/20) are

reinserted. Transformation in ccdb resistant strain (OneShot CCDB survival cells) using standard protocol and 5 ul

of CPEC reaction product.

On plates with colonies are always blue colonies as well as white ones -> no information on assembly success.

CCDB screening and test

Five white colonies from pRB12 were picked as well as a blue colony as a control. Conlony PCR using intron iTaq

polymerase was performed and 6x 0.5 mL LB+Cm were inoculated with the six colonies.

Screening is positive -> miniprep of colonies #3 and #4 from 2 ml liquid culture. Purified plasmid pRB12 was transformed to TOP10, DH5alpha and OneShot on LB+Cm to check CCDB functionality. OneShot should grow whereas plates of DH5alpha and TOP10 were expected to stay empty since ccdb causes cell death.

All strains grow on LB+Cm which shows that ccdb doesn't work as expected. ccdb with RBS was amplified from pDONR

plasmid.

Results and Discussion

Assembly and/or Transformation were inefficient, blue colonies on each plate suggest that a lot of template was in

the Transformation mix. The positive control doesn't provide information on whether the assembly was successful.

CCDB screenig of pRB12 was positive but transformation in DH5alpha, TOP10 and OneShot ccdb survival cells showed

that ccdb is unfunctional. In the following, all the insert from pDONR will be used including a promoter and the

ccda gene. We will work with indC on pSB1C3 with PPTases on separate plasmids to be able to easily test different

combinations of T-Domain and PPTase.

===CPEC Conditions Assay (Ralf)===

We performed CPEC assembly of pRB13 with eight different CPEC conditions to see whether we get difeerent results.

Table 11.1 CPEC mix for assembly of pRB13

Table 11.2 CPEC Assembly of pRB13

100 ul of competent TOP10 were transformed with 5 ul of reaction product and incubated for 30 hours at 37 °C.

Apparently there is no big difference using various CPEC conditions. We will continue with the standard protocol.

PPTase test (Ralf)

pRB3-10 are plasmids containing indC (pRB-3-6) or bpsA(pMM64) (pRB7-10) and a PPTase (sfp, svp, svp(pMM65), entD).

This week the blue colonies are screened and sequenced for validation.

colony PCR screening #1

Two blue colonies are picked from each plate and screened with the primers of the PPTase they should have and the

ones which they could have if something went wrong. PPTase PCR products werde used as a positive control and TOP10

without any plasmid as a negative control. So numbers 5 to 18 are subdivided into alpha and beta.

The gel is not conclusive. Therefore I will repeat the screening but screen every colony for all PPTases.

MiniPrep of pRB4-10

5 ml liquid cultures of TOP10+pRB4-10 have been miniprepped using QIAquick miniprep kit. Only blue cultures have

been prepped (except pRB9 because there was no blue culture). pRB4-6 and pRB10 were prepped in duplicates (liquid

cultures of two different clones) whereas there are only single preps of pRB7-9. This is because pRB3-6 contain

indc ahich we will use for further studies. pRB6 and 10 contain entD, which is the E. coli endogenous PPTase and

was previously declared to be "inefficient" in activating bpsA [Takahashi 2007].

Concentrations of the prepped plasmids have been measured using ??ThermoScientific NanoDrop

Minipreps were reduced to a concentration of about 50 ng/ ul for sequencing and 1 ng/ ul for further studies.

MiniPrep PCR screening #2

pRB9 is negative. All screens are positive in the svp(pMM65) and entD section. In entD this could be due to genomic

DNA in the miniprep. svp(pMM65) is also positive in all plasmids, which is strange. Maybe the PCR conditions have

been too tolerant. Nevertheless the bands of the plasmids which should carry the respective PPTase appear to be

stronger. We will sequence the plasmids anyways.

Sequencing of pRB3-10

plasmid minipreps have been sequenced by GATC [reference] using the sequencing primers VF2 and VR. The sequencing

results show that all the inserts are correct except for pRB9, which isn't a pity because we have previously shown

with pKH1-2 that the combination of bpsA(pMM64) and svp(pMM65) successful produces indigoidine.

In the following pRB4-beta, pRB5-alpha, pRB5-beta, pRB6-alpha and pRB10-alpha have been selected so that there are

single versions of pRB3-10 for furhter experiments.

Glycerols stocks pRB3-10

Preparation for T-Domain exchanges

We assemble pRB14, which is a version of pRB3, in which the T-Domain is exchanged by the whole insert of the pDONR vector. Since pRB13 grew on DH5alpha and TOP10 we hope that pRB14 won't. This vector will be used to exchange T-Domains and will be modified to get a version without the PPTase sfp and the cutting sites EcoRI and SpeI in indC. To check whether a successfully exchanged T-Domain can be activated by a defined PPTase, we build pSB2K3-derived plasmids with a single PPTase (sfp, svp, entD or delC) under control of the lac-Promotor and the RBS BBa_B0029.

Fragment Amplification

PCR of Fragments for PPTase plasmids

Table 12.x PCR for pRB15-18: 25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul.

pSB2K3 contains the 3'-part of the reversed Primer in an internal lac-Operator-structure, so the PCR product is just half of the backbone (2500 bp instead of 4800 bp). We will use the same primers but pSB3K3 of the 2013 spring distribution plate 5 well 5E; which is around 3000 bp.

Table 12.x PCR for pRB15-18: 25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul.

We get unspecific product -> repetition with more stringent conditions

PCR of Fragments for T-Domain exchange

Table 12.x PCR for pRB13: 25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul.

Gel Extraction

Gel extraction was performed using QIAquick gel extraction kit. DNA yield was measured using NanoDrop Spectrophotometer ND-1000

Table 12.x DNA concentrations for assembly of pRB-T

CPEC Assembly and Transformation

pRB14-18

Table 12.x

CPEC Assembly pRB14-18

Transforation according to standard protocol for chemical transformation

• TOP10 with pRB15-18 (-> LB+Kan)
• TOP10 with pRB15-18 and pMM64, respectively (-> LB+Kan+Amp)
• TOP10 with pMM64 (-> LB+Amp)
• OneShot with pRB14 (-> LB+Cm)

• OneShot+pRB14: small colonies after 20 hours
• TOP10+pRB15-18: colonies on all plates
• TOP10+pMM64+pRB15-18: (almost) no colonies after 20 hours; hopefully due to indigoidine growth retardation
• TOP10+pMM64 TraFo was efficient

We will perform PCR screening in triplicates of TOP10+pRB15-19 and OneShot+pRB14 to get a first impression on whether the assembly was successful.

Plasmid Validation

PCR Screening

We use forward primers of the insert and standard reversed primer VR to screen pRB14-18 in triplicates. We use iTaq DNA-polymerase in 20 ul PCR mix:

• 10 ul iTaq 2x Master Mix
• 2 ul Primer 10 uM each
• 6 ul water
• colony pick from plate

Table 12.x

PCR screening pRB14-18

Annealing temperature according to NEB Tm calculator for Taq DNA polymerase and VF2

Test Transformation

OneShot and TOP10-sells have been transformed with pRB14. We expect OneShot cells to grow as usual and no colonies on TOP10. As a control group we transformed OneShot as well as TOP10 with pRB3.

pRB19

pRB19 is a pRB14-derived plasmid without the PPTase sfp. The genotype is pSB1C3-lacPromotor-BBa_B0034-indCdT(ccdB)

Table 12.x

PCR Amplification of pRB14 for pRB19

Smear -> Annealing temperatures in the touchdown-part were too high, so RB21 3' end couldn't bind properly. Second run with milder conditions.

Table 12.x

PCR Amplification of pRB14 for pRB19 #2

Smear -> we will run another strategy, i.e. amplification of indC from pRB14 with RB27/46 to further assemble it with pSB1C3(RB21/22)

Table 12.x

PCR Amplification of indC(RB27/46) for pRB19 #3

Gel extraction was performed using QIAquick gel extraction kit. DNA yield was measured using NanoDrop Spectrophotometer ND-1000

Table 12.x DNA concentrations for assembly of pRB19

Table 12.x

CPEC Assembly pRB19

Transformation into OneShot Survival cells. Doing colony PCR for screening with primer KH9/VR which would give a fragment size of around 1.8 kbp: (Note: Throw picked colony 4 away since unintendently pooled 2 colonies in PCR tube). Used iTaq.

Table 12.x

PCR screening pRB19 and pRB21

colony PCR for pRB19 and pRB21

Colony PCR for pRB19 gave a lot of unspecific bands, probably because of less stringent parameters. Oculutated 6 ml of TB+Cm with colony 2 for MP preparation.

Biobricks

The PPTases sfp, svp, entD and delC and the indigoidine synthetase indC will be prepared for submission to the registry. indC contains two RFC10-cutting site, that have been removed during the assembly of pRB22.

pRB21 series

pRB21 is a pSB1C3-plasmid with lacPromotor, BBa_B0034 and indC. After the first assembly, EcoRI and SpeI cutting sites in indC will be removed to yield pRB22. After cutting site removal, the native T-Domain will be exchanged with ccdB to yield pRB22. pRB23 has the same genotype as pRB20 but with two PCR steps less.

Fragment Amplification

Table 12.x

PCR Amplification of indC for pRB21 #1

Amplification was not successful. Next try from PCR product with short primers as a rePCR.

Table 12.x

PCR Amplification of indC for pRB21 #2

Heidelberg 20130815 PPTBBa indCpRB21 cut2.PNG

Gel Extraction

Gel extraction was performed using QIAquick gel extraction kit. DNA yield was measured using NanoDrop Spectrophotometer ND-1000

Table 12.x DNA concentrations for assembly of pRB21

CPEC Assembly and Transformation

Table 12.x

CPEC Assembly pRB21

Validation

Colony PCR for pRB21 was unsuccessful (see gel picture), try again with less strigent parameters and positive control: KH5/VR on MP pRB3, which should give an amplicon size of around 2 kbp.

Table 12.x

PCR screening pRB21

colony PCR forpRB21

Colony PCR for pRB21 worked now as well as the positive control. Oculutated 6 ml of TB+Cm with colony 8 for MP preparation.

PPTase BioBricks

have to look up used primer/template combinations again Konrad (talk)

Table 12.x

PCR Amplification of PPTases for BioBrick submission

Heidelberg 20130815 PPTBBa indCpRB21 cut2.PNG

Gel Extraction

Assembly and Transformation

Digest

Ligation

Preparation for T-Domain exchange

Fragment Amplification

PCR #1 for fragment for T-Domain exchange

No band at 6400 bp. Second try with less stringent conditions

PCR #2 for fragment for T-Domain exchange

Still nothing; maybe the MiniPrep didn't work so fine. We will screen pRB19-transformed cells on pRB19. We want to get a first impression on whether the Domain-exchange works, so we'll use pRB14 instead.

PCR #3 for fragment for T-Domain exchange

PCR worked but with very low yield. We try to amplify from pRB19-transformed cells instead of the miniprep.

25 ul Phusion HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

PCR #1 for TTE-Domains

bpsA-TTE and tycC6-TTE worked, the other ones will be amplified using less stringent conditions

25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

PCR #2 for TTE-Domains

delH5-TTE worked, though with low yield. entF-TTE will be amplified in a third PCR.

25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

PCR #3 for TTE-Domains

entF-TTE didn't work. We will postpone this PCR.

T-Domain exchange

CPEC Assembly and Transformation

Transformation in TOP10 according to standard protocol. Incubation for 30 hours at 37 °C and 10 hours

on the bench.

BioBricks

Fragment Preparation

25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

PCR #1 for fragment for T-Domain exchange

PCR worked well. To make sure that we don't transform template plasmid pRB21, we digest the pRB22d(EcoRI-SpeI) fragment Gel Extraction together with pRB22d(SpeI-EcoRI) with DpnI for four hours at 37 °C.

The Digest was purified using QiaGen Nucleotide Removal Kit. Unfortunately I dropped 2/3 onto the table... The final DNA concentration was 47.5 ng/ ul according to NanoVue. Standard CPEC assembly was performed in the old broken BioRAD cycler which didn't cool down to 53 °C but only to 58 °C for the annealing step. Hopefully it worked. TOP10 cells were transformed with 5 ul of CPEC master mix.

Transformation was successful. Ten colonies were screened using primers VF2 and KH6 in a 20 ul iTaq Master Mix according to standard protocol (50 °C).

Probes number x to x were purified using Qiaquick PCR purification kit and digested with NEB DpnI, EcoRI-HF and SpeI-HF over night to see whether pRB22 is really free of those restriction sites. The result was put on an AgaroseGel.

Lanes 1 to 4 show a slight smear beneath 200 bp. Lane 5 (probe 10) doesn't show that smear and the

specific band runs maybe 300 bp higher. So maybe the first four lanes contained restriction sites and

the last one didn't. We put probes 1 and 10 in 40 ml culture for midiprep. The midiprep will then be

digested as well so that we get certainity about the restriction sites. In parallel we did a colony PCR from probe 10 with KH3/4 using Phusion Flash HF to already have our

fragment for pRB23 for the T-Domain-exchange.

Preparation for T-Domain exchange

Assembly of pRB19

PCR indC(ccdB) RB27/28 and pSB1C3 RB21/22 25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

Gel Extraction with QIAquick gel extraction kit.

Digest ran for 1 hour at 37 °C, was put on a agarose gel, gel extracted and eluted in 15 ul water.

Concentration was measured using NanoVue: 30 ng/ ul. 10 ul were put together with 10 ul of Phusion

Flash HF Master Mix for standard CPEC assembly. OneShot and TOP10 cellc were transformed using different amounts of CPEC product.

Ten colonies were screened using iTaq with KH9/VR and RB35/VR, respectively, to see whether the assembly worked and sfp was kicked out.

The screening was positive, so probe 10 was kept for 10 ml liquid culture (TB+Cm). Miniprep of 4 ml yielded 69.4 ng/ ul in 50 ul. Miniprep was used for PCR with primers KH3/4 to get the fragment pRB19 without ccdB for insertion of T-Domains via CPEC. First we tried two different polymerases at two different conditions each. The first is Phusion Flash

HF 2x Master Mix (ThermoScientific) and the other is Phusion HF 2x Master Mix (NEB).

Gel extraction and measurement using NanvoVue. The final table of T-domain concentration is as

follows:

Assembly of pRB15-18

pRB15-18 were unable to be sequenced with various primers. We now use pSB3K3 backbone from plate 3 of the 2013 spring distribution and assemble those PPTase-plasmids de novo.

PCR was run with with a fragment of the Tyrocidine group with slightly suboptimal conditions. It will be repeated using otimal conditions.

Gel extraction yielded 144.2 ng/ ul (Nanodrop)

CPEC was performed using pSB3K3 gel extraction and gel extractions made for assembly of pRB3-10. TOP10 cells were transformed using standard protocol.

Cells were plated on LB+Kanamycin and incubated at 37 °C for 24 hours.

Five colonies of each plate were screened using iTaq polymerase and two primer combinations for each colony. This is VF2/PPTase_rv primer and PPTase_fw/VR primer.

pRB16 is obviously wrong. We checked the PCR product we used for the assembly and recognized that it's wrong, so we have to amplify it again from S. verticillus. DelC screenings show another small band, we have too check whether the primers bind elsewhere on the plasmid and run a negative control with those primers.

Assembly of pKH4

pKH4 is a pRB3 derived version of an indigoidine synthetase expression plasmid w/o sfp as activating PPtases. So the two plasmid strategy can be conducted (at first) with this plasmid instead to rely on pRB21. Basically the idea is to use restriction sites before and after sfp to discard it and to religate with another sequence to yield a functional backbone.

pRB3 has a BamHI before and a NheI cutting site after sfp. pMM64 and pMM65 both can be cut with a combination of BglII and XbaI to yield for example a 164 bp long fragment with compatible ends to the linearized pRB3. This fragment unfortunately contains a T7lac hybrid promoter which could interfere with our used primers, but still is worth a shot.

• digest mix volume: 30 µl (3 µl NEBuffer 3.1, 6.5 µl pMM65 (~481 ng), 0.3 µl (BglII, XbaI) each, 19.9 µl H2O)

Resulting gel of digestion of pMM65 with BglII and XbaI in order to gain sequence with cohesive ends to linerized pRB3 w/o sfp.

• digestion gave expected bands at 2 kbp and 2.4 kbp but band at ~160 bp is missing. Reaction was carried out with not enough DNA.
• prepared ON for pMM64/65 MP as well as pRB3 MP
• the next day ON were extended to 2x 4 ml LB cultures which were then

prepped after several hours of incubation at 37 °C

• MP with cultures: DNA conc. measurement with NanoVue gave: 280

ng/µl (pMM64), 225 ng/µl (pMM65), 250 ng/µl (pRB3); analysis gel shows different concentration for pMM65 (~15 ng/µl ?)

Analytical gel for verification of MP pMM64/65 and pRB3: concentrations measured with NanoVue seem to be ok for pMM64 and pRB3, but much less for pMM65. 2log ladder lane contain 420 ng each; 100 V, TAE

• prepare digestion (20 µl), 37 °C, 1.5 h:
  • pMM64: 2 µl NEBuffer 3.1, 17 µl pMM64, 0.5 µl (BglII, XbaI)
  • pRB3: 2 µl NEBuffer CutSmart, 10 µl pRB3, 0.5 µl (BamHI, NheI), 7

µl H2O

Digestion of pMM64 (BglII, XbaI) and pRB3 (BamHI, NheI) were desired bands for insert of ~170 bp and backbone-indC of ~6300 bp were cut out. 100 V, TAE

• after gelextraction (elution in 20 µl) ligation was conducted with 2

µl 10x T4 ligase buffer, 1 µl T4 ligase, 2 µl pRB3 Dig_GE, 8 µl pMM64 Dig_GE and 7 µl H2O for 40 min at RT

Analytical gel of 1 µl of ligation mix, compared to 0.5 µl of pRB3 digestion with (BamHI, NheI). Additionally 1 µl of MP pMM65 was put on gel to verify previous concentration measurement. 1.5 µl of log2 ladder, 100 V, TAE

• transformation in TOP10 with 1 µl and 5 µl of ligation mix

pRB15-18

The first mini

Screening delC

Screening pKH4

A miniprep of probe 4 was sent to sequencing, which was positive.

Assembly of pKH4-derivates

pKH4 is used for assembly of pKH5 and variants of pKH4 with all the different T-Domains.

KH3/4-PCRs were unsuccessful, so we did a gradient PCR

We run the same protocol for 3x 50 ul.

nothing

16 x 10 ul with Q5 and Tm calculator suggested parameters (63 °C annealing), pRB19 ligation as template

We tried NEB Phusion HF with 61 °C annealing temperature in a 10-, 20- and 50 ul reaction from pRB19 ligation and in a 20 ul reaction from pKH4 ind T100 (left). As well we ran with Phusion HF (59 °C annealing) in 20 ul reactions with 0, 1 and 2 ul DMSO (Biorad Cycler 2)

For further evaluation of the single plasmid strategy we admired a plasmid with bpsA instead of indC as indigoidine synthetase. For a quick shot we wanted to use pKH4 and pRB7 for a restriction approach with the enzymes KpnI and BamHI.

• prepare digestion (20 µl), 37 °C, ~0.5 h:
  • pKH4: 2 µl NEBuffer CutSmart, 4 µl pKH4, 13 µl H2O, 0.5 µl (KpnI, BamHI)
  • pRB7: 2 µl NEBuffer CutSmart, 2.3 µl pRB7, 14.7 µl H2O, 0.5 µl (KpnI, BamHI)

Digestion of pKH4 (KpnI, BamHI) and pRB7 (KpnI, BamHI). Desired bands for insert of ~3.8 kbp and backbone+sfp of ~3.9 kbp were expected. 100 V, 0.8 % Agarose, TAE

The restriction for pKH4 did not work correctly due to the fact that with assembly of pKH4 the BamHI cutting site was removed and the plasmid was only linearized by the KpnI cutting site. For pRB7 two bands at almost equal site can be expected and thereby the digestion approach can not be used in this manner. We will try again with a similar approach like for the assembly of pKH4 as here a nonsense sequence instead of the PPtases will be introduced into pRB7.

• preparation of pRB7 MP by retransformation of TOP10 (as no glycerol stock is available).

Amplification of svp

The svp fragment we used so far was wrong, so we try to amplify it again from the genome, using various primers:

• RB13/14
• RB15/16
• RB29/30
• RB39/40

Gradient on biometra cycler (72 °C, 65 °C, 58 °C)

svp was always right; only after assembly it turned wrong. We will try to bring it in using restriction cloning and to sequence it; maybe the screening is just inconclusive.

Amplification of indC-fragments

indC fragments are amplified from Plum genome and indC short PCR product in a 50 ul Phusioin HF reaction. The fragments are:

• RB27/69
• RB70/71
• RB68/46
• RB68/KH4
• KH5/RB46
• RB27/46 from pRB14
• RB27/28