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Revision as of 21:48, 2 October 2013

Contents 1 Tyrocidine-Indigoidine-Fusion - Cloning & Validation 1.1 Gibson Assembly 1.1.1 pPW03 (Phe-Ind) 1.1.2 pPW04 (Asn-Ind) 1.1.3 pPW05 (Val-Ind) 1.2 Transformation 1.3 Restriction Digest 1.3.1 Procedure 1.3.2 Results 1.3.3 Discussion 1.4 Sequencing & Transformation into BAP-I 1.5 Screening PCR for positive BAP-I-colonies 1.5.1 Procedure 1.5.2 Results 1.6 Induction, SDS-PAGE & TLC 1.6.1 Procedure 1.6.2 Results 1.6.3 Discussion

Tyrocidine-Indigoidine-Fusion - Cloning & Validation

Gibson Assembly

Gibson assemblies were prepared according to the following formulas:

pPW03 (Phe-Ind)

fragment	concentration [ng/µl]	volume for gibson assembly [µL]
A	12	0.89
B	8	4.29
C	6	2.86
D	35	1.96

pPW04 (Asn-Ind)

fragment	concentration [ng/µl]	volume for gibson assembly [µL]
D	35	3.87
E	20	1.06
F	20	5.08

pPW05 (Val-Ind)

fragment	concentration [ng/µl]	volume for gibson assembly [µL]
D	35	3.45
G	15	1.26
H	16	3.78
I	20	1.51

Transformation

The constructs prepared by gibson assembly were chemically transformed (heat shock) into competent DH10ß.

Restriction Digest

Procedure

DH10ß - colonies were picked and grown overnight in LB with Chloramphenicol. After a miniprep, a restriction digest with EcoRI was performed, the expected bands were:

• pPW03 (Phe-Ind): 5858 and 3183
• pPW04 (Asn-Ind): 5858 and 3582
• pPW05 (Val-Ind): 5858 and 3105

Results

All samples showed the expected bands.

BAP 12 Eco.png pPW03 and pPW04 digested with EcoRI, samples A, B, C & D for pPW03 and B, C & D for pPW04 show the expected bands.

BAP 3 Eco BAP TetI EcoPst.png pPW05 digested with EcoRI, all samples show the expected bands. The six lanes on the left are neglectable.

Discussion

Clones pPW03 A, B, C & D; pPW04 B, C & D; pPW05 A, B, C, D, E & F show the expected bands, hence two of each clone were sent to sequencing.

Sequencing & Transformation into BAP-I

Vectors were sequenced with the Primers used for the assembly. All sequences turned out to be positive, hence the minipreps that were sent to sequencing, were used for chemical transformation into BAP-I-cells accoding to the standard protocol. For each transformation 10µl and the rest were plated seperately. After less then two days, blue colonies occured on all plates, hence colony-PCR were performed on 5 colonies of each plate (3 blue ones and 2 white ones).

In parallel, BAP-I-cells were transformed with the remaining Gibson-Mix for pPW03, pPW04 and pPW05. Cells were picked and grown in LB with Chloramphenicol overnight. After miniprep, restricion digest with EcoRI was performed in 20 µl volume. Again, the expected bands were:

• pPW03 (Phe-Ind): 5858 and 3183
• pPW04 (Asn-Ind): 5858 and 3582
• pPW05 (Val-Ind): 5858 and 3105

300px

As these results were not as clear as for the sequenced samples, we did not continue work on the direct BAP-transformants.

Screening PCR for positive BAP-I-colonies

Procedure

In order to screen for positive BAP-I-colonies, blue and white colonies were picked and used in colony-PCR.

Results

COLPCR 03 04 05 BAP Tyc.png Screening for the Tyc-part in blue (B1, B2, B3) and white (W1, W2) colonies from different plates with cells that were transformed with pPW03, pPW04 or pPW05

COLPCR 03 04 05 BAP Ind.png Screening for the Tyc-part in blue (B1, B2, B3) and white (W1, W2) colonies from different plates with cells that were transformed with pPW03, pPW04 or pPW05 (left of black line) and screening for the Ind-part (right of black line)

Almost all colonies were positive for both modules, except the colonies that were transformed with pPW03 (Phe-Ind). For these constructs, the bands that show up do not match the expected bands:

• expected: xxxx
• obtained: yyyy

Induction, SDS-PAGE & TLC

Procedure

Cells were grown at 37°C and induced at OD600 = ~0.5 with 1mM IPTG. Cultures that were transformed with pPW05 turned blue after about 30 to 60 minutes. Cells were kept at 30°C overnight and the synthetized NRP was extracted and purified from samples. Herefore, 1ml culture was centrifuged at 14,000 rpm for 15 minutes, supernatant was kept for further analysis. The pellet was washed in 1ml Methanol and centrifuged at 14,000 rpm for 15 minutes. Supernatant was kept for further analysis and pellet was resuspended in 500µl DMSO. The crude supernatant, the methanol-wash and the purified NRP were used for TLC. As a control, purified indigoidine (treated with the same protocol) and its washes were used.

As solvent, a mixture of 95% Dichloromethane and 5% Methanol was used. However, all samples were running at the solvent front, hence a less polar solvent, i.e. 100% Dichloromethane was used, and qualitatively better results were obtained.

For the SDS-PAGE, remaining culture was treated as described previously and prepared for SDS-PAGE. Not only pPW05-cells, but also pPW03 and pPW04-positive cells were tested, in order to check for proper expression of the NRPSs.

Results

TLC 1.png first TLC - one biological replicate for Val-Ind and one biological replicate for Indigoidine, including the crude supernatants and methanol-washes were used.	TLC 2.png second TLC	TLC 3.png third TLC	TLC 4.png fourth TLC
TLC 5.png fifth TLC - showing the running behavior for three biological replicates of Val-Ind and four technical replicates of Indigoidine. Samples were applied alternatingly in order to exclude the possibility of different running behavior due to placement on the TLC.	SDS 3rd.png SDS-PAGE, stained with Coomassie. The bands for the expected Val-Ind-NRPS are visible as indicated by the blue arrows. The golden arrows mark highly probable bands for the other NRPSs that are however qualitatively not 100% clear due to e.g. smear.

Discussion

Especially the last TLC-picture nicely shows that the indigoidine-control has a significantly and reproducibly different sunning behavior on the TLC. Different biological replicates (different clones) were used for Valin-Indigoidine and in all cases, it is running lower than the control - as expected.

As far as the SDS-PAGE is concerned, the expected protein sizes are:

• Phe-Ind-NRPS: 247 kDa
• Asn-Ind-NRPS: 261 kDa
• Val-Ind-NRPS: 242 kDa
• Note: the indigoidine-synthetase alone: 145 kDa

As indicated by the blue arrows, the Val-Ind-NRPS is clearly present in all of the tested samples. For the other two NRPSs, there are bands at the expected height, as indicated by the golden arrows, these are however qualitatively not as good as the ones for the Val-Ind-NRPS due to smear or low expression.