Team:Freiburg/Notebook/19 August

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Meeting

attendants: Jakob, Julia, Manuel, Rüdiger, Sandra, Sophie, Theo
Time: 9:00 - 10:00

green light receptor

already done:

• Ccas some colonies (check if positive)
• CcaR still doesn't work

To-do:

• cph8 still won't work, we should ask to get the original sequence

blue light receptor

already done:

• receptor and NOT-Gate assembly

To-do:

• add Promotor-RBS and terminator

red light receptor

already done:

• Promotor-RBS-ho1-terminator ready
• Promotor-RBS-pcyA-terminator ready

To-do:

• receptor still doesn't work (not possible to amplify via a PCR)

Lysis cassette

already done:

• quick change with the 11 bases didn't work
• did a 3A assembly with the single parts

To-do:

Precipitator

already done:

• finally the genes arrived
• GST from the bioss toolkit can't be amplified from the supported vector, Rüdiger should do a positive control next time, if it still doesn't work he should get new primer pairs

To-do:

• PBD + inducible promotor

other stuff

• Hauke Busch recommended CellDesigner to Rüdiger to do the modelling, Rüdiger will download it and try to install it
• modelling should be done for the precipitator, escpecially the Ni-Histidin-binding and for the plastic binding domain, maybe also the GST-Tag affinity
• Tobi will organize moving the stuff out of the lab after the wiki freeze
• Sandra will ask for the appointment to talk at the MPI

green light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

blue light receptor

Sandra, Sophie: primer design for NOT-gate to get NEH I restriction sites inside instead of Spe I. We can not cut with Spe because our trp-promoter contains Spe restriction sites.

red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

Lysis cassette

2A Assembly Sequencing

Investigators:Theo

results came in: Only RBS to be seen! :( 

So for the next week I had to do the whole 2A assembly from the beginning (including waiting 1 day for the S15 stock to grow in order to prep).

IMPORTANT: be careful of ligations in 2A assemblies, use little vector and try to stay between 1:1 and 1:3 vector:insert ratios... The reason this didn't work is that I used a whole lot of the vector. Notice: little vector means 20ng

Precipitator

3A-assembly

Digestion

Procedure

1. add H₂O (38μl-DNA )
2. 5 μl NEB4 buffer (stored at iGEM’s, -20°C)
3. 5 μl 10x BSA (used 1:10 diluted sample stored at iGEM’s, -20°C)
4. DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
5. 1 μl restriction enzymes (stored at iGEM’s, -20°C)
6. heat for 1-2 hours 37°C (6 hours if time)
7. heat for 20 minutes 80°C (inactivation of enzymes)
8. keep at 4°C if you cannot continue

Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:

Restriction enzymes you need to cut the vector, insert1 and insert 2:

1.Digestion

Investigators: Rüdiger

digested with EcoRI and PstI.

Gel extraction kit

Investigators: Theo

DNA fragments of the digestion( see above) were excised from the gel.

Ligation

Investigators: Rüdiger

Trying to ligate the precipitator (excised from the gel) in the pSB1C3 vector. Ratio of ligation: 1:3