Team:Heidelberg/Tyrocidine week21 interms

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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.

Colony PCRs. On the left: ccdB (all positive except 8). On the right: Indigoidine (all positive).

Digests of ccdB-indC constructs 1-5 with Pst1 and EcoRI.

Digests of ccdB-indC constructs 6-10 with Pst1 and EcoRI.

Digests of ccdB-indC3 and 7 with Pst1 and EcoRI. Results were positive.

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.

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