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From 2013.igem.org

Contents 1 Tyrocidine-Indigoidine-fusion - standardization 1.1 Reassembly 1.1.1 pPW03 (Phe-Ind) 1.1.2 pPW04 (Asn-Ind) 1.1.3 pPW05 (Val-Ind) 1.1.4 Results 1.1.5 Optimization 1.2 Validation 1.2.1 Procedure 1.2.2 Results

Tyrocidine-Indigoidine-fusion - standardization

Reassembly

As CPEC-approaches did not work out so far, a reassembly of the fusion constructs was run in parallel. Herefore, a standardized indC-module, that was prepared by the Indigoidine group via CPEC, was used.

The following formulas for gibson assembly were calculated:

pPW03 (Phe-Ind)

Fragment	Concentration [ng/µl]	Volume for gibson assembly [µL]
A	12	3.29
B	8	3.95
C	6	2.63
D*	120	0.13

pPW04 (Asn-Ind)

Fragment	Concentration [ng/µl]	Volume for gibson assembly [µL]
D*	120	0.25
E	20	3.75
F	20	6.00

pPW05 (Val-Ind)

fragment	Concentration [ng/µl]	Volume for gibson assembly [µL]
D*	120	0.26
G	15	4.21
H	16	3.95
I	20	1.58

Results

COLPCR 1' 2' 3' Tyc.png Screening for the Tyrocidine-part in the various colonies picked after electroporation of the Gibson-Mix.

COLPCR 1' 2' 3' Ind.png Screening for the Indigoidinde-part in the various colonies picked after electroporation of the Gibson-Mix.

COLPCR 1' 2' 3' Ind REPEAT.png Repetition of the sceening with overnight-cultures.

As shown in the images above, this approach did not lead to the desired result, as mostly colonies that were negative in the colony PCR were growing on the plate. Therefore, we decided to change the Gibson-protocol once more, using less backbone and more indC. For this optimization, we focussed on the Val-Ind-NRPS encoded by pPW05. However, the only positive colony (pPW05* B) was kept for further analysis.

Optimization

With the following optimization:

Fragment	Concentration [ng/µl]	Volume for gibson assembly [µL]
D*	120	1.67
G	15	1.33
H	16	5.00
I	20	2.00

Results: Only few colonies were growing after electroporation.

Validation

Procedure

Restriction digests were performed with the so far promising samples. The gels after digest (which were run at 135V) all had strange running patterns, as it seemed as if all of the samples ran too high.

RESTR ECO BGLHIND 3.png Restriciton digest with EcoRI and BglII&HindIII

RESTR pPW03 1 2 ECOPST MFE ECOHIND.png Restriction digest with EcoRI&PstI, MfeI and EcoRI&HindIII

RESTR pPW03 1 2 ECOPST NOT PCR.png Restriction digest with EcoRI&PstI and NotI, PCR of the insert

Even though the sequencing was positive for all of those samples, we were concerned because of the unexpected restriction patterns. Therefore, we picked 12 colonies from remaining plates, let them grew overnight, took 2ml for miniprep and induced the remaining 1ml. In addition, we transformed the promising samples, that were already positive in sequencing, into BAP-I. Transformation was also performed for TOP10 and NEB-Turbo cells, which were also co-transformed with a plasmid carrying a PPTase and kanamycin resistance. Instead of plating them and picking colonies, we decided to grow the transformants directly in LB+Cm-selection medium. The next morning, we induced them as well.

Results

There were 7 out of the 12 colonies that turned blue after induction with 1mM IPTG, hence they were miniprepped and digested with EcoRI and PstI. Only half of the volume was loaded on a gel, the other half was purified with isopropanol.

RESTR ECOPST pPW05'2-10 PARTS.png Restriction digest with EcoRI and PstI, ran at 135V

RESTR ECOPST pPW05'2-10 PARTS CLEAN.png Restriction digest purified with isopropanol, ran at 100V

Again, the pattern of bands was uncommon and unexpected, hence we purified the mixture to remove residual enzyme and cut DNA with isopropanol. Analysing this on a gel that was run at 100V yielded a better result. The obtained bands met our expectations. For validation, a TLC was run with the samples, which again showed the slower migration of Val-Ind. Finally, we could submit our samples to the registry, as sequencing was positive for all linkers.