Team:TU Delft/Notebook/cloning-plan

From 2010.igem.org

(Difference between revisions)
(Two-way ligations)
(Two-way ligations strategies)
Line 4: Line 4:
==Two-way ligations strategies==
==Two-way ligations strategies==
 +
By using two-way ligaiton we ligate two parts to each other. The backbone of the smallest part was cut open. The largest part was cut out of its backbone. To prevent self-ligation we cut the backbone with an additional restriction enzyme.
 +
 +
In the case that the smallest part was the second part we used cloning strategy A
 +
 +
The smallest part was cut with EcoRI and XbaI
 +
 +
The largest part was cut with EcoRI and SpeI
 +
 +
It is important that the extra enzyme only cuts in the backbone and not in the part itself
 +
[[Image:TU Delft 2 way ligation A.tif]]
[[Image:TU Delft 2 way ligation A.tif]]
 +
 +
In the case that the smallest part was the first part we used cloning strategy B
 +
 +
The smallest part was cut with SpeI and PstI
 +
 +
The largest part was cut with XbaI, PstI
 +
 +
It is important that the extra enzyme only cuts in the backbone and not in the part itself
[[Image:TU Delft 2 way ligation B.png]]
[[Image:TU Delft 2 way ligation B.png]]
 +
 +
In the end all BioBricks have to be cloned in the pSB1C3 plasmid backbone. With our strategy all the BioBricks are still in its original plasmid backbone, which in most cases was pSB1A2. We performed a pSB1C3 migration which is illustrated by cloning strategy C. 

Revision as of 06:48, 18 October 2010

Contents

Cloning Plan

Once we decided what we wanted to achieve and with which genes, we designed the following cloning plan.

Two-way ligations strategies

By using two-way ligaiton we ligate two parts to each other. The backbone of the smallest part was cut open. The largest part was cut out of its backbone. To prevent self-ligation we cut the backbone with an additional restriction enzyme.

In the case that the smallest part was the second part we used cloning strategy A

The smallest part was cut with EcoRI and XbaI

The largest part was cut with EcoRI and SpeI

It is important that the extra enzyme only cuts in the backbone and not in the part itself


TU Delft 2 way ligation A.tif


In the case that the smallest part was the first part we used cloning strategy B

The smallest part was cut with SpeI and PstI

The largest part was cut with XbaI, PstI

It is important that the extra enzyme only cuts in the backbone and not in the part itself


TU Delft 2 way ligation B.png


In the end all BioBricks have to be cloned in the pSB1C3 plasmid backbone. With our strategy all the BioBricks are still in its original plasmid backbone, which in most cases was pSB1A2. We performed a pSB1C3 migration which is illustrated by cloning strategy C.


TU Delft 2 way ligation C.png

Our plan

Alkane Degradation

Hydrocarbon & Salt Tolerance

Hydrocarbon Solubility

Sensing

RBS Characterizaton