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From 2013.igem.org

Contents 1 Konrad 1.1 validation of ccdB constructs 2 Construction of ccdb-constructs pRB11-13 2.1 amplification of CPEC fragments 2.2 Agarose Gel and Gel Extraction 2.3 CPEC assembly and Transformation 2.4 CCDB screening and test 3 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)

component	concentration	amount
Phusion Flash HF MM	2x	25 ul
Primer each	10 uM	5 ul
water		14.7 ul
template	>20 ng/ul	0.3 ul

NI13/14 in T100
cycles	temperature (°C)	time (s)
1	98	10
8	98	1
	64 ↓ 0.5	5
	72	10
22	98	1
	65	5
	72	10
1	72	60
1	12	-
KH1/2 in biometra
cycles	temperature (°C)	time (s)
1	98	10
8	98	1
	56 ↓ 0.5	5
	72	10
22	98	1
	65	5
	72	10
1	72	60
1	12	-
KH3/4 in T100
cycles	temperature (°C)	time (s)
1	98	10
10	98	1
	62 ↓ 0.5	5
	72	120
20	98	1
	57	5
	72	120
1	72	300
1	12	-
NI15/16 in T100
cycles	temperature (°C)	time (s)
1	98	10
30	98	1
	65	5
	72	120
1	72	300
1	12	-
KH5/6 in C1000
cycles	temperature (°C)	time (s)
1	98	10
10	98	1
	63 ↓ 0.5	5
	72	10
30	98	1
	59	5
	72	10
1	72	30
1	12	-
NI17/18 in C1000
cycles	temperature (°C)	time (s)
1	98	10
10	98	1
	63 ↓ 0.5	5
	72	10
30	98	1
	65	5
	72	10
1	72	30
1	12	-
KH7/8 and NI19/20 in BioRad old one
cycles	temperature (°C)	time (s)
1	98	10
30	98	1
	65	5
	72	10
1	72	30
1	12	-

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.

1 ug 2-log; KH3/4; KH5/6; KH7/8; NI15/16; NI17/18; NI19/20

1 ug 2-log; KH3/4; KH5/6; KH7/8; NI15/16; NI17/18; NI19/20

1 ug 2-log; NI15/16

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.

CPEC assembly Phusion Flash HF in biometra cycler
cycles	temp (°C)	time (s)
1	98	10
10	98	1
	55	5
	72	120
1	72	300
1	12	-

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.

ccdb screening pRB12 colonies with NI13/14 in
cycles	temperature (°C)	time (s)
1	98	10
35	98	60
	60	30
	72	80
1	72	300
1	12	-

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.