Team:LMU-Munich/Weekly Journal

September

24-28 September 2012

Now, the Sporo vector is cloned into pSB_Bs4S.

We all are busy setting up our website and finding the best ways to display our results!

Sitting on the computer every day and analyzing the sheer number of microscopy pictures...

17-21 September 2012

Last microscopy experiments with the clean deletion mutants. They are glowing even brighter!

Finished cloning of mKate2 constructs. Now the evaluation of this reporter BioBrick with three different promoters, P_liaI, P_lepA and the Anderson promoter J23101, can start.

Finished cloning of Sporo vector in pSB1C3.

Evaluation of pSB_Bs0K-P_spac with lacZ

Inverter measured quantitavely via ONPG assay

For our Suicide switch, the first platereader measurement of Bacillus subtilis strain W168 containing thrC::PspoIVB-ECF41 (through transformation with pSBBs4S-PspoIVB-ECF41) and sacA::pydfG-luxABCDE (through transformation with pSBBs3C-luxABCDE-PydfG).

Knockouts: One last run of the germination assay on our triple and quadruple mutants. All results of these assays can be found compiled on our data page.

10-14 September 2012

To clean up our Sporobeads from vegetative B. subtilis cells we tried three different methods: French Press, sonification and lysozymes. A great difference was observed after the treatment with lysozyme! Furthermore the lysozyme did not damage our fusion protein as GFP fluorescence was still obtained in microscopy!

For the Suicide switch, PspoIVB-ECF41 in pSB_BS4S, PydfG/PsspK/PspoIVB in pSB_BS3C-luxABCDE brought into B. subtilis

More work on the knockouts: We were really surprised at our great results so far, and repeated the assay once again.

3-7 September 2012

All of our CotZ-GFP variants were examined with fluorescence microscopy! The brightest spores derived from our P_cotyz-cotZrep-gfp-terminator mutants!

We worked more on the knockouts aspect of the GerminationSTOP module. We ran another germination assay for our triple and quadruple mutants. Plates of our spores diluted at 10^-2, 10^-4 and 10^-6 show NO GERMINATION for our triple and quadruple mutants, and plenty of germination for the WT168 positive control! We will try plating undiluted mutant spores to see if any germination occurs.

More work on the Suicide switch! PsspK, PspoIVB and PydfG cloned into pSB1C3 and verified by sequencing.
PspoIVB-ECF41 cloned into pSB_BS4S and verified by sequencing.
PydfG/PsspK/PspoIVB brought into pSB_BS3C-luxABCDE

lacZ was succesfully cloned into pSB_Bs4S-P_Xyl and is functional (blue colonies with IPTG and X-Gal)

A collection of useful tags in Freiburg standard and with RBS included was cloned into pSB1C3. The tags are: 3xFlag, HA, cMyc, 10xHis and Streptavidin.

August

26-31 August 2012

Finally, we got our first glowing spores!! After 4 months of hard work we have the first proof that this module works.

More on germination knockouts -- we created quadruple mutants using two variations on past mutants: cwlD::kan + cwlJ::spec + gerD::cm + sleB::mls and gerD::cm + sleB::mls + cwlJ::spec + cwlD::kan. See our Strains Collection. Germination assay was performed on triple and quadruple mutants.

For the Suicide switch, MazF and ECF41 cloned into pSB1C3 and verified by sequencing

The BioBrick lacZ for B. subtilis was shown to be functional in pSB_Bs0K-P_spac in E. coli and B. subtilis. (blue color on plates with IPTG and X-Gal)

The genes luc+ and mKate2, synthesized by GeneArt were successfully cloned into pSB1C3 and sequenced.

20-24 August 2012

Inverter with lacZa was finished and works qualitatively.

Good and bad news this week. First the good one: Another big step towards the GFP-Sporobeads is done! We have the CotZ constructs in pSB_BS1C! Hopefully it will integrate easily! :D Now the bad one: Finally we could start with the P_cgeA evaluation! But contrary to our expectations this promoter did not show any activity.

13-17 August 2012

For the knockouts, we used last week's double mutants to create triple mutants as follows: cwlD::kan + sleB::mls + cwlJ::spec ; cwlD::kan + sleB::mls + gerD::cm ; cwlD::kan + cwlJ::spec + gerD::cm ; gerD::cm + sleB::mls + cwlJ::spec. See our Strains Collection.

ß-glactosidase assay of the Anderson promoters J23100, J23102, J23103 in pSB_Bs1C-lacZ in B. subtilis. The xylose-inducible promoter with the according repressor (which has a constitutive promoter, RBS and terminator) xylR-P_Xyl was cloned into pSB1C3 and sequenced.

At last Bacillus was keen on integrating the pSB_BS3C-luxABCDE-P_cgeA!! And the clean deletions of CotZ and CgeA worked out, too!

6-10 August 2012

Germination knockouts: We created four double-mutants using resistance-cassettes to knock out germination genes as follows: cwlD::kan + sleB::mls ; gerD::cm + sleB::mls ; gerD::cm + cwlD::kan ; cwlJ::spec + cwlD::kan. See our Strains Collection. Of these double mutants, we tested three strains in our germination assay. We also created the resistance cassette knockout cwlB::kan.

Finally, the last PstI site could be removed and pSB_Bs3C-luxABCDE was completed. Also, the double terminator B0014 was cloned into pSB1C3.

The final Promoter-CotZ-GFP-Terminator constructs in pSB1C3 are ready!! And Promoter-CgeAmut constructs are finally finished too.

July

30 July - 3 August 2012

GFP-Terminator is ready for use and the AgeI site in CgeA is mutated!

23-27 July 2012

This week is our Human Practice week! From 23^rd to 25^th of July we enjoyed the CAS SynBio conference and the visit of 10 iGEM teams! This weekend, high school students participated in our Students Practical Course for three days and developed their ideas for useful Sporobeads! It was a great week!

Plate reader measurements of the Bacillus promoters P_liaG, P_liaI and P_lepA finished. Look at Data!

16-20 July 2012

Not many of us are working on our modules this week, as we are helping out for CAS SynBio conference and organising the Students Practical Course.

Plate reader measurements of the Anderson promoters J23100, J23101, J23102, J23103, J23106, J23107, J23113, J23114, J23115, J23117, J23118 in the Bacillus reporter vector pSB_Bs3C-luxABCDE completed. Look at Data Anderson promoters!

Cloning of the Anderson promoters J23100, J23102, J23103, J23106 in the reporter vector pSB_Bs1C-lacZ for β-galactosidase assays finished.

9-13 July 2012

The vectors pSB_Bs4S-P_Xyl , pSB_Bs1C-lacZ and pSB_Bs4S were succesfully completed and tested by restriction digest as well as red colony color.

We started with the clean deletions of CgeA and CotZ this week, seems like it will take for ever if you judge from the protocol length... What took even longer is the pSB_BS3C-luxABCDE-P_cgeA, but it is finished now!

2-6 July 2012

In the knockouts part of the GerminationSTOP module, clean deletions of germination genes sleB and cwlB from PCR were accomplished. DNA purified and frozen to be later transformed with Bacillus.

β-galactosidase assays of the promoters P_liaG, P_liaI, P_veg are finished. Look at Data!

Cloning of the Anderson promoters J23100, J23101, J23102, J23103, J23106, J23107, J23113, J23114, J23115, J23117, J23118 in pSB1C3 finished.

Our first plate reader experiments with P_cotyz and P_cotv are running! Like expected they both show activity in the late stationary phase, thus during sporulation!

June

25-29 June 2012

pSB_BS3C-luxABCDE-P_cotyz and pSB_BS3C-luxABCDE-P_cotv integrated into the B. subtilis genome! Bad luck, we found an additional AgeI site in cgeA, seems like this is the reason why we could not fuse gfp to it... Mutagenesis primers are designed and ordered!

18-22 June 2012

CotZ and GFP constructs are finished, now we can try to fuse them together!

11-15 June 2012

Knockouts: Clean deletions of germination genes cwlD, cwlJ, and gerD from PCR accomplished. DNA purified and frozen to be later transformed with Bacillus. Edit: the clean deletions were never used for transformation, but remain ready for future use.

4-8 June 2012

Still working on the Promoter-Gene and now GFP-Terminator constructs!

May

28 May-1 June 2012

GFP in BioBrick standard is readyyyy!

21-25 May 2012

pSB_BS3C-luxABCDE-P_cotyz and pSB_BS3C-luxABCDE-P_cotv are ready for transformation into Bacillus!

Promoters P_liaG, P_liaI, P_veg and P_lepA are now in the vector pSB1C3 as BioBrick standard for the registry.

The Anderson promoters J23100, J23101, J23102, J23103, J23106, J23107, J23113, J23114, J23115, J23117, J23118 are now in the Bacillus reporter vector pSB_Bs3C-luxABCDE for measuring their activity as luminescence.

The Bacillus promoters P_liaG, P_liaI and P_lepA are in the Bacillus reporter vector pSB_Bs3C-luxABCDE.

14-18 May 2012

Combining the BioBricks and integrating them into different vectors works good! But still there are constructs missing, we'll keep on working! The fusion of the up and down fragment of CotZ finally works!

7-11 May 2012

The last weeks we tried to fuse the up and down fragment of our spore crust gene together for its clean deletion. For CgeA we have the first fused fragments! CotZ needs a little more attention :)

Cloning of P_liaG, P_liaI, P_veg and in vector pSB_Bs1C-lacZ finished.

April

30 April-4 May 2012

We have the first essential BioBricks for this module: pSB1C3-CotZ, pSB1C3-P_cotV, pSB1C3-P_cotYZ, pSB1C3-P_cgeA, pSB1C3-CgeA! Still working on the pSB1C3-GFP.

23-27 April 2012

pSB_Bs3C-luxABCDE with still one PstI site was created with RFP in the multile cloning site to have a vector for promoter measurments. edit: This PstI site was removed later and only that backbone is submitted to the registry.

16-20 April 2012

The Sporobead team starts the work in the lab!

The cloning of the reporter vector pSB_Bs1C-lacZ </b> was finished.

9-13 April 2012

We created the single mutant cwlD::kan.

2-6 April 2012

In our germination genes knockout work, we successfully created our first single knockouts of germination genes using a resistance cassettes: sleB::mls, gerD::cm and cwlJ::spec. See our Strains Collection. We tried knocking out cwlB using the kan resistance cassette. Mutants of cwlB::kan grew very poorly.

With pSB_Bs0K-P_spac our first vector for our Bacillus BioBrick Box was completed.

March

26-30 March 2012

Knockouts: We tried knocking out cwlB using the tet resistance cassette. Mutants of cwlB::tet grew very poorly.

19-23 March 2012

For our germination gene knockouts, we decided which genes to knock out for the germination stop. Based on the work of J. Kim and W. Schumann (2009), we decided to knock out genes cwlB, gerD, cwlJ, and sleB. From the research of B. Setlow et al (2001), we also chose cwlD.

February

20-24 February 2012

Team fully formed!

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