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: File:Lys17-P81208-2.gb :( 

what is to be seen is that the lysis genes are indeed cloned behind the RBS but because (as our instructor told us) the vector containing RBS was not treated with antarctic phosphatase, the small DNA part between SpeI and PstI digestion sites managed to wedge itself back in...

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... Notice: little vector means 20ng

Also, use phosphatase for 2A assemblies!!!

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