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Team:SDU-Denmark/Notebook
From 2009.igem.org
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Project task-management notebook
Todo
Create biobrick RIP in registry
Create biobrick RIP+Export in registry
Doing
Plasmid backbone
Task: Find a backbone for both e.coli and b. subtilis
People: Helle and Mike
July 7th 2009:
We found the backbone, [http://partsregistry.org/wiki/index.php/Part:BBa_I742123 BBa_I742123], which other teams have found to be compatible with both both gram positive and gram negative bacteria, such as e. coli.
After talking to HQ, it turns out that the quality of their stock is bad, and they will try to get some home from older teams. Furthermore, it might simply be better to use a more specifik backbone for e. coli and for b. subtilis.
(mike)
July 8th 2009:
We decided to start working with [http://partsregistry.org/Part:pSB1A3 pSB1A3] instead, which is a high-output e. coli backbone.
Output: 100-300 number pr. cell. Resistance: ampicillin (we're going with 50 µg/mL first)
(mike)
Inducible promoter
Task: Find a promoter, that can be induced
People: Anna
July 7th 2009
I found the inducible promoter BBa_R0011 that is regulated by LacI.
It is a strong promoter, that will be on in stains without LacI.
(anna)
E. coli strain
Task: Find a e.coli strain
People:
July 7th 2009
The plasmid seems to be working in most types (no need for ccdB) Jacob suggests E. coli KG22, which can turns on the operon by adding IPTG.
July 8th 2009
We'll use Top10 e.coli strain
Promoter for e.coli
Task: Find a constitutive and inducible promoter
People: Anna and Kir
July 7th 2009
Constitutive promoter
We'll use [http://partsregistry.org/Part:BBa_J23100 BBa_J23100] as constitutive promoter. Its located in QC09 Kit Plate 1, Well C18.
Inducible promoter
We'll use [http://partsregistry.org/Part:BBa_R0011 BBa_R0011] as an inducible promoter. It's located in: QC09 Kit Plate 1, Well 6G, Plasmid: pSB1A2
Use:
To induce this promoter region, generally add 1mM of IPTG to a plate (amount will vary by cell strain). That is, total volume of stuff in plate (usually around 0.025 liters) * 0.001 = X amount of liters that you will add to the solution.
RBS
Task: Find a RBS
People: John and Julius
July 7th 2009
We'll use [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 BBa_B0034]. The sequence is: 5'-AAAGAGGAGAAA-3'. It's located in: Kit plate 1, well 2M.
Binding to the site is not regulated, and it has been reported to be very effective.
Terminator
Task: Find a terminator
People: Marc
July 7th 2009
We'll use [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 BBa_B0015]. This is a double terminator consisting of BBa_B0010 and BBa_B0012. This is the most commonly used terminator. It seems to be reliable. Note, however, that BBa_B0014 is a better part for forward and reverse termination.
It's located in: QC09 Plate 669, Well 41811.
