Team:Freiburg/Notebook/29 July

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Contents 1 Meeting 1.1 green light receptor 1.2 blue light receptor 1.3 red light receptor 1.4 Lysis cassette 1.5 Precipitator 1.6 other stuff 2 blue light receptor 2.1 Glycerol stocks of Not-Gate 3 Lysis cassette 3.1 Sequencing of Lysis Cassette V.2 (Quickchange-modified K098995 + Lysis genes K124017) 4 Precipitator

Meeting

attendants: Julia, Manuel, Rüdiger, Sandra, Theo, Tobi Time: 9:00 - 11:00

green light receptor

already done:

• Ccas has to sites where we need to do a mutagenesis (quick change), both were done seperately, but the no colonies showed up (wrong PCR program)
• CcaR has one site where we need to do a mutagenesis (quick change), this has been done, colonies were growing

To-do:

• miniprep, digestion and sequencing of the CcaR clones
• do the Ccas PCR again
• next steps would be the big assembly wiht pcyA

blue light receptor

already done:

• PCR of Lov-tap and Not-gate. Not-gate sequencing was good. PCR of Lov-tap did not work the first time and the second time with the temperature gradient PCR

To-do:

• creating new primer for the Lov-tap as the old ones had a high probabylity for secondary structures

red light receptor

already done:

• waiting for cph8 from Mexico
• for pcyA we did a new transformation from the original plasmid

To-do:

• over night culture, miniprep and stock from the pcyA plates
• 3-A-Assembly of pcyA and Terminator

Lysis cassette

already done:

• the quick change didn't work the first time (wrong template DNA)
• second time went well, but the transformation was difficult (only 10 colonies)

To-do:

• do the miniprep and the sequencing

Precipitator

already done:

• GFP-PDB: cloning doesn't work only a few colonies

To-do:

• ask again if the gene synthesis is done: Rüdiger
• Gibson cloning with the GST-Tag, TEV-site and Promotor-RBS

other stuff

• deadline for the parts: 10th of september
• name all the files you upload on the wiki with the prefix Freiburg2011_
• start documenting the lab stuff in the wiki
• still missing: logo and text from hiss, MPI, Serva, Invitrogen
• Modelling: Rüdiger will ask Simon if he can help us along
• sponsoring give-aways: key tag, postcards, movie, pencils
• Lab tracks: Tobi wants to try it
• cooperation with Upsala: Yeahhh!

blue light receptor

Glycerol stocks of Not-Gate

Investigators: Sandra

M45d was sequenced and we did glycerol stocks of bacteria containing M45d.

Lysis cassette

Sequencing of Lysis Cassette V.2 (Quickchange-modified K098995 + Lysis genes K124017)

Test Sequencing showed that the parts were assembled correctly.

File #1: You can see the EcoRI, XbaI sites and also the Quickchange-modified K098995 as well as the XbaI-SpeI SCAR and the start of the Lysis genes K124017

File #2: You can see part of the Lysis genes K124017 and the SpeI and PstI sites

Right-click to download the annotated ape (.gb) file #1

Right-click to download the annotated ape (.gb) file #2

What can immediately be noticed is the absence of a RBS (ribosome binding site) between the lamba cI regulated Promotor (R0051 of K098995) and the holin ATG of K124017. This had skipped our attention at the planing phase and was a source of headaches during the next days... :)

Precipitator

Testdigest

Name: Ruediger	Date: 29.07
Continue from Experiment (Date) Miniprep 28.07 (Name)
Project Name: GFP Pbd

For one reaction you need: For Mastermix: 12 Number of samples+2extra

4μl	H2O	84
1μl	Buffer, NEB4	14
1μl	BSA (10x)	14
0,5 μl	Enzym 1	7
0,5 μl	Enzym 2	7
3 μl	DNA	1

10 μl total volume

Give 3 μl of DNA in an eppi and add 7μl of the mastermix.

Incubate for about 1h at 37°C.

Add 1 μl Loading dye buffer and load the gel.

Take a picture of the gel, print picture and label the lanes!

nothing worked, no GFP inserts seens anywhere. Gel picture was not saved by machine. -Sequencing: no GFP in constructs - retry by Theo.