Team:Heidelberg/Tyrocidine week21 interms

From 2013.igem.org

ccdb Construct

Amplification

ccdb-C(tycC2)-indC plasmid (pJS01) Amplification of C(tycC2), ccdB and pSB1C3 DNA fragments (indC was stored in the fridge from former experiments).

ccdB pcr contents

What	µl
primer_fw (PW38)	2
primer_rv (PW41)	2
template (Gateway pDONR vector)	2
Phusion Flash 2x Master Mix	10
ddH20	4

PCR-Program ccdB sequence

Cycles	Temperature [°C]	Time [min:s]
1	98	0:10
8	98	0:01
	70 ↓0.5	0:05
	72	0:15
28	98	0:01
	65	0:05
	72	0:15
1	72	1:30
1	10	inf

pSB1C3 PCR contents

wWhat	µl
primer_fw (PW23)	2
primer_rv (PW42)	2
template (pSB1C3)	0.5
Phusion Flash 2x Master Mix	10
ddH20	5.5

PCR-Program pSB1C3 sequence

Cycles	Temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	60	0:20
	72	1:00
1	72	10:00
1	10	inf

C(TycC2) PCR contents

What	µl
primer_fw(PW21)	2
primer_rv(PW14)	2
template (B. Parabrevis)	1
Phusion Flash 2x Master Mix	10
ddH20	5

PCR-Program C(TycC2)

Cycles	Temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:01
	59	0:05
	72	1:00
1	72	5:00
1	10	inf

After amplification, an analytical gel electrophoresis was run to quantify DNA concentrations.

Check on PCR-products by gel eletrophoresis. All products showed an appropriate size.

C(TycC2) PCR contents

What	Size [bp]	Estimated concentration
pSB1C3	2500	35
ccdB	600	30
C(TycC2)	1000	30
indC	4000	100

Assembly

Gibson assembly was followed by purification with isopropanol and transformation by heat shock into ccdB resistent and non-resistent (Top10) cells.

After 10 hours of culture growth, colony PCRs and test digests with Pst1 and EcoRI were conducted. Afterwards, minipreps of cultures containing the plasmids and concentration measurements were performed accompanied by test digests. Finally, positive constructs were transformed again and prepared for sequencing.

ccdB nanodrop measurements

Colony	Concentration [ng/µl]
ccdB1	863.5
ccdB2	436.9
ccdB3	1258.5
ccdB4	260.5
ccdB5	106.6
ccdB6	1064.4
ccdB7	1213.0
ccdB8	1301.4
3B	982.3
3*B	982.4

Sequencing results of ccdB3 and ccdB7 (just partwise sequencing) were positive. Biobrick can be submitted.