Team:SDU-Denmark/Notebook

=Project task-management notebook=

Find Plasmid backbone
Task: Find a backbone for both e.coli and b. subtilis

People: Helle and Mike

July 7th 2009: We found the backbone, 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 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)

E. coli strain
Task: Find a e.coli strain

People: Mike

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 first.

Promoter for e.coli
Task: Find a constitutive and inducible promoter

People: Anna and Kir

July 7th 2009

Constitutive promoter

We'll use BBa_J23100 as constitutive promoter. Its located in QC09 Kit Plate 1, Well C18.

Inducible promoter

We'll use 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 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 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.