"

From 2013.igem.org

Contents 1 RFC10 Standardization of Tyc Modules 1.1 Sequencing 2 Tyrocidine-Indigoidine-fusion - standardization 2.1 Amplification 2.2 Results 2.3 CPEC 2.4 Validation 2.5 Parallel-approach: Reassembly

RFC10 Standardization of Tyc Modules

Sequencing

Primer	OK?	Comments	File
Tyc AdCom
VR_rv		-	File:Heidelberg RFC10 TycASeq.zip
VF2_fw		-	File:Heidelberg RFC10 TycASeq.zip
Tyc B1
VR_rv		silent mutations: 4595 GCC (Ala) to GCG (Ala); 4598 CTT (Leu) to CTG (Leu)	File:Heidelberg RFC10 TycB1Seq.zip
VF2_fw			File:Heidelberg RFC10 TycB1Seq.zip
Tyc C5
VR_rv		backbone was ok, wrong insert; since the insert was amplified with the same protocol like TycB1 we think that the primers were mixed up	File:Heidelberg RFC10 TycC5Seq.zip
VF2_fw		backbone was ok, wrong insert; since the insert was amplified with the same protocol like TycB1 we think that the primers were mixed up	File:Heidelberg RFC10 TycC5Seq.zip
Tyc C6
VR_rv			File:Heidelberg RFC10 TycC6Seq.zip
VF2_fw		silent mutations: 4595 GTA (Val) to GTG (Val)	File:Heidelberg RFC10 TycC6Seq.zip

Tyrocidine-Indigoidine-fusion - standardization

So far, the Tyrocidine-Indigoidine-fusion constructs had two illegal cutting sites of RFC-10 restriction enzymes. Hence, we tried a CPEC-approach for which we reamplified a fragment from the vector with primers that introduced a mutation at the desired position.

Amplification

what	µl
pPW05 (dil.)	1
RB68	2
RB69	2
Phusion Flash 2x Master Mix	10
ddH20	5

Cycles	temperature [°C]	Time [min:s]
1	98	0:05
35	98	0:05
	59	0:10
	72	3:00
1	72	10:00
1	10	inf

Results

CPEC

After gel-extraction of the large fragment, the following procedure was followed:

• 22µl H2O, 20µl of the large fragment, 2µl of the short fragment, 1µl DpnI and 5µl 10x CutSmart buffer were incubated for 2 hours at 37°C
• Mixture was purified with isoprop, washed with ethanol and eluted in 10µl H2O
• 10µl Phuson Flash Master Mix was added and the followin protocol was run:

Cycles	temperature [°C]	Time [min:s]
1	98	0:10
5	98	0:01
	53	0:05
	72	3:00
1	72	10:00
1	10	inf

Validation

In order to validate colonies that were transformed with the CPEC-Mix, Restriction digest and sequencing was performed. As the unwanted restriction sites should have been mutated in the positive samples, using EcoRI as restriction enzyme should yield a single band at the size of the linearized vector (9049bp for pPW03, 9448bp for pPW04 and 8971bp for pPW05). Restriction digest was conducted with the standard protocol in a 20µl reaction volume.

The image on the lefthand-side shows the gel after 20 minutes at 100V, as this did not lead to good separation, the gel was let run for another 20 minutes. The gel after 40 minutes is displayed on the right side.

The sample for pPW01 and samples 1 and 3 for pPW05 were sent to sequencing as they migrated at the expected size.

Parallel-approach: Reassembly

In order to have a second pillar to lean on, we decided to reamplify fragment D (indC) without the two restriction restriction sites. The template for this was created by the Indigoidine-group which had used the CPEC-approach described above.

what	µl
indC*	0.5
PW15	2
PW16	2
Phusion Flash 2x Master mix	10
ddH20	5.5

Cycles	temperature [°C]	Time [s]
1	98	5
12	98	5
	61	5
	72	100
23	98	5
	58	10
	72	100
1	72	600
1	10	inf

We obtained (as expected) clearly visible, bright bands at the expected fragment size (~4000bp)

ples to neonate screening.