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From 2013.igem.org

Contents 1 RFC10 Standardization of Tyc Modules 1.1 Ligation of digested Fragments each with linearized pSB1C3 1.1.1 DNA Concentration of Digestions 1.1.2 Reaction 1.2 Transformation (Standard Protocol) into TOP10 1.3 Picking & Screening I 1.3.1 Colony PCR with Amplification Primers 1.3.2 Screening Colony PCR with VR_rv and VF2_fw 1.4 Picking & Screening II 1.4.1 Screening PCR 2 Tyrocidine-Indigoidine-Fusion - Standardization of Constructs 2.1 Amplification 2.2 Results 2.3 CPEC

RFC10 Standardization of Tyc Modules

Ligation of digested Fragments each with linearized pSB1C3

DNA Concentration of Digestions

Fragment	Source	Protocol	Concentration
Tyc AdCom	AT_01/AT_02	Reamplification 2013-08-31xxx	5.0 ng/µl
Tyc B1	AT_09/AT_10	Reamplification 2013-08-31xxx	22.5 ng/µl
Tyc C5	AT_11/AT_06	Reamplification 2013-08-31xxx	19.2 ng/µl
Tyc C6	AT_07/AT_08	Reamplification 2013-08-31xxx	35.7 ng/µl
pSB1C3	mediprep of ONC	-	14.4 ng/µl

Reaction

1:1 Ratio of pSB1C3:insert

Fragment	Size [kb]	Concentration [ng/µL]	Amount for Reaction [µL]	10x T4 DNA Lig Buffer [µL]	T4 DNA Ligase [µL]	+ddH2O
add Backbone pSB1C3; 4.2 kb; 14.4 ng/µl			3.5
Tyc AdCom	3.2	5.0 ng/µl	7.6	2	1	5.9
Tyc B1	3.2	22.5 ng/µl	1.7			11.8
Tyc C5	3.2	19.2 ng/µl	2			11.5
Tyc C6	3.9	35.7 ng/µl	1.5			12

Transformation (Standard Protocol) into TOP10

2 plates per Ligation one with less one with more transformed cells.

Picking & Screening I

picked red clones Picked 3 clones per plate= 6 clones per construct

Colony PCR with Amplification Primers

Phusion Flash Colony PCR (see amplification protocols above)

Result NO Product?!

Screening Colony PCR with VR_rv and VF2_fw

iTaq Colony PCR Result NO Product?!

Picking & Screening II

NEGATIVE RESULTS BEFORE BECAUSE: mrfp was replaced by insert thus the positive colonies are WHITE!!! Picked white colonies

Screening PCR

Screened all picked colonies of one fragment: positive Screened one clone per fragment: positive

Tyrocidine-Indigoidine-Fusion - Standardization of Constructs

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

Unfortunately the efficiency of this was not high enough, as no colonies were visible on the plates after heat-shock transformation in BAP-I cells. The Amplification and CPEC will be repeated next week.