Team:Heidelberg/Tyrocidine week19 biobrickmod



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


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 15.9
Tyc B1 3.2 22.5 ng/µl1.711.8
Tyc C5 3.2 19.2 ng/µl211.5
Tyc C6 3.9 35.7 ng/µl1.512

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.


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


Heidelberg PCR 1' 2' 3'.png


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.