Difference between revisions of "Team:UBonn HBRS/Description/B Subtilis"

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== General ==
 +
''Bacillus subtilis'' is a gram-positive bacterium which can be found almost everywhere, especially
 +
soil. It can form a protective endospore to resist extreme environmental conditions.
 +
 
 +
We decided to work in this organism because ''Bacillus subtilis'' is a bacteria, which secretes proteins
 +
and enzymes naturally to its surrounding medium. <br />
 +
The goal was to utilise this attribute for enzyme production and create a “cell factory”. Instead of
 +
lysating bacteria, it was desired to have a continuous production of enzymes in a liquid culture that
 +
we can extract from the supernatant. <br />
 +
Repeated inoculations of new bacteria cultures are not necessary because they will stay in the
 +
stationary phase. Only the media has to get replaced.
 +
 
 +
''Bacillus subtilis'' is classified as a GRAS-organism (general regarded as safe) by the U. S. Food &
 +
Drug administration1 and widely used in S1 laboratories as a model organism. It is also present in
 +
economy as probiotics in pharmacies or fungicide in agriculture. <br />
 +
Furthermore biotechnological industry already uses ''Bacillus subtilis'' to synthesize some enzymes.
 +
 
 +
=== Usage ===
 +
For using our product we have different approaches. <br />
 +
Either just using the supernatant to deink our paper fibers or make the time intensive and expensive
 +
step of purificating our enzymes and then proceed with deinking the paper. For purificating we
 +
attached a His-tag on our enzymes and separate them from other components of the supernatant
 +
with nickel columns.
 +
 
 +
In order to reach the project goals plasmids containing the needed genes had to be transformed into
 +
the bacteria. <br />
 +
We worked with two different ''Bacillus subtilis'' strains we got donated by the department of
 +
Pharmaceutical Biotechnology of the Greifswald University, Germany. <br />
 +
We got the wild type strain ATCC 6051 and also the promisingly LS8P-D strain. The LS8P-D stain
 +
is a derivate from the protease-deficient WB800 strain, where two additionally genes got deleted.
 +
The lack of proteases is essential to secrete our enzymes without damaging them. <br />
 +
After trying out multiple protocols we decided to accomplish transformation by electroporation.
 +
The principle of electroporation relies on creating small holes in the cell wall of bacteria, by
 +
sending a current through them. The plasmids enter the bacteria through the holes before the
 +
organism repair them.
 +
 
 +
The iGEM Team Freiburg and the iGEM Team Bonn collaborated in order to get working
 +
transformation protocols for Bacillus subtilis. The resulting manual “How to ''Bacillus subtilis''” can
 +
be found on our <br />
 +
<html><a class="btn btn-default" href="//2016.igem.org/Team:UBonn_HBRS/Collaborations">Collaborations Page</a></html>
 +
 
 
{{UBonn_HBRS/footer}}
 
{{UBonn_HBRS/footer}}

Revision as of 02:37, 19 October 2016

 

B. Subtilis

General

Bacillus subtilis is a gram-positive bacterium which can be found almost everywhere, especially soil. It can form a protective endospore to resist extreme environmental conditions.

We decided to work in this organism because Bacillus subtilis is a bacteria, which secretes proteins and enzymes naturally to its surrounding medium.
The goal was to utilise this attribute for enzyme production and create a “cell factory”. Instead of lysating bacteria, it was desired to have a continuous production of enzymes in a liquid culture that we can extract from the supernatant.
Repeated inoculations of new bacteria cultures are not necessary because they will stay in the stationary phase. Only the media has to get replaced.

Bacillus subtilis is classified as a GRAS-organism (general regarded as safe) by the U. S. Food & Drug administration1 and widely used in S1 laboratories as a model organism. It is also present in economy as probiotics in pharmacies or fungicide in agriculture.
Furthermore biotechnological industry already uses Bacillus subtilis to synthesize some enzymes.

Usage

For using our product we have different approaches.
Either just using the supernatant to deink our paper fibers or make the time intensive and expensive step of purificating our enzymes and then proceed with deinking the paper. For purificating we attached a His-tag on our enzymes and separate them from other components of the supernatant with nickel columns.

In order to reach the project goals plasmids containing the needed genes had to be transformed into the bacteria.
We worked with two different Bacillus subtilis strains we got donated by the department of Pharmaceutical Biotechnology of the Greifswald University, Germany.
We got the wild type strain ATCC 6051 and also the promisingly LS8P-D strain. The LS8P-D stain is a derivate from the protease-deficient WB800 strain, where two additionally genes got deleted. The lack of proteases is essential to secrete our enzymes without damaging them.
After trying out multiple protocols we decided to accomplish transformation by electroporation. The principle of electroporation relies on creating small holes in the cell wall of bacteria, by sending a current through them. The plasmids enter the bacteria through the holes before the organism repair them.

The iGEM Team Freiburg and the iGEM Team Bonn collaborated in order to get working transformation protocols for Bacillus subtilis. The resulting manual “How to Bacillus subtilis” can be found on our
Collaborations Page

© iGEM Team Bonn 2016 Top ↑