Team:LMU-Munich/Weekly Journal

September

24-28 September 2012

Now, the Sporo vector is cloned into [http://partsregistry.org/Part:BBa_K823022 pSB_Bs4S] and we are trying to insert [http://partsregistry.org/Part:BBa_K823019 lacZ].

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 [http://partsregistry.org/Part:BBa_K823029 mKate2] constructs. Now the evaluation of this reporter BioBrick with three different promoters, [http://partsregistry.org/Part:BBa_K823001 P_liaI], [http://partsregistry.org/Part:BBa_K823002 P_lepA] and the Anderson promoter [http://partsregistry.org/Part:BBa_K823005 J23101], can start.

Finished cloning of Sporo vector in pSB1C3.

Evaluation of [http://partsregistry.org/Part:BBa_K823026 pSB_Bs0K-P_spac] with [http://partsregistry.org/Part:BBa_K823019 lacZ].

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K823040 Inverter] measured quantitively via β-Galactosidase assay.

For our Suicide switch, the first plate reader measurement of Bacillus subtilis strain W168 containing thrC::P_spoIVB-ecf41_{Bli aa 1-204} (through transformation with pSBBs4S-P_spoIVB-ecf41_{Bli aa 1-204}) and sacA::P_ydfG-luxABCDE (through transformation with pSBBs3C-luxABCDE-P_ydfG) was performed.

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!

P_lepA was fused into the finished vector [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823025 pSB_BS3C-luxABCDE] to evaluate this BioBrick vector and compare in to the version where there is still one forbidden restriction site in it.

For the Suicide switch, P_spoIVB-ecf41_{Bli aa 1-204} in pSB_BS4S, and P_ydfG/P_sspK/P_spoIVB in pSB_BS3C-luxABCDE were brought into B. subtilis.

More work on the knockouts: We were really surprised at our great results so far, and repeated the germination assay on our triple and quadruple mutants 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 mutants, and plenty of germination for the WT168 positive control! We will try plating undiluted mutant spores to see if any germination occurs at higher concentrations.

More work on the Suicide switch! [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823042 P_sspK], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823048 P_spoIVB] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823041 P_ydfG] were cloned into pSB1C3 and verified by sequencing.
P_spoIVB-ecf41_{Bli aa1-204} were cloned into pSB_BS4S and verified by sequencing.
P_ydfG/P_sspK/P_spoIVB were brought into pSB_BS3C-luxABCDE.

[http://partsregistry.org/Part:BBa_K823019 lacZ] was succesfully cloned into [http://partsregistry.org/Part:BBa_K823024 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: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823034 3xFlag], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823035 HA], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823036 cMyc], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823037 10xHis] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823038 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, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823044 MazF] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823043 Ecf41_{Bli aa 1-204}] were cloned into pSB1C3 and verified by sequencing.

The BioBrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823019 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823028 luc+] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823029 mKate2], synthesized by GeneArt, were successfully cloned into pSB1C3 and sequenced.

20-24 August 2012

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K823040 Inverter] with lacZα was finished and it works qualitatively.

Good and bad news this week. First the good news: Another big step towards the GFP-Sporobeads is done! We have the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823049 CotZ constructs] in pSB_BS1C! Hopefully it will integrate easily! :D Now the bad news: 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 ; and gerD::cm + sleB::mls + cwlJ::spec. See our Strains Collection.

ß-glactosidase assay of the Anderson promoters [http://partsregistry.org/Part:BBa_K823004 J23100], [http://partsregistry.org/Part:BBa_K823006 J23102], [http://partsregistry.org/Part:BBa_K823007 J23103] in pSB_Bs1C-lacZ in B. subtilis was performed. The xylose-inducible promoter with the according repressor (which has a constitutive promoter, RBS and terminator) [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823015 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 [http://partsregistry.org/Part:BBa_K823025 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!! Our 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 the 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 [http://partsregistry.org/Part:BBa_K823000 P_liaG], [http://partsregistry.org/Part:BBa_K823001 P_liaI] and [http://partsregistry.org/Part:BBa_K823002 P_lepA] are 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 the CAS SynBio conference and organising the Students Practical Course.

Plate reader measurements of the Anderson promoters [http://partsregistry.org/Part:BBa_K823004 J23100], [http://partsregistry.org/Part:BBa_K823005 J23101], [http://partsregistry.org/Part:BBa_K823006 J23102], [http://partsregistry.org/Part:BBa_K823007 J23103], [http://partsregistry.org/Part:BBa_K823008 J23106], [http://partsregistry.org/Part:BBa_K823009 J23107], [http://partsregistry.org/Part:BBa_K823010 J23113], [http://partsregistry.org/Part:BBa_K823011 J23114], [http://partsregistry.org/Part:BBa_K823012 J23115], [http://partsregistry.org/Part:BBa_K823013 J23117], [http://partsregistry.org/Part:BBa_K823014 J23118] in the Bacillus reporter vector pSB_Bs3C-luxABCDE completed. Look at Data Anderson promoters!

Cloning of the Anderson promoters [http://partsregistry.org/Part:BBa_K823004 J23100], [http://partsregistry.org/Part:BBa_K823006 J23102], [http://partsregistry.org/Part:BBa_K823007 J23103], [http://partsregistry.org/Part:BBa_K823008 J23106] in the reporter vector pSB_Bs1C-lacZ for β-galactosidase assays finished.

9-13 July 2012

The vectors [http://partsregistry.org/Part:BBa_K823024 pSB_Bs4S-P_Xyl ], [http://partsregistry.org/Part:BBa_K823021 pSB_Bs1C-lacZ] and [http://partsregistry.org/Part:BBa_K823022 pSB_Bs4S ] were succesfully completed and tested by restriction digest as well as checked for red colony color.

We started with the clean deletions of CgeA and CotZ this week. It seems like it will take forever 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 was purified and frozen to be later transformed with Bacillus.

β-galactosidase assays of the promoters [http://partsregistry.org/Part:BBa_K823000 P_liaG], [http://partsregistry.org/Part:BBa_K823001 P_liaI], [http://partsregistry.org/Part:BBa_K823003 P_veg] are finished. Look at Data!

Cloning of the Anderson promoters [http://partsregistry.org/Part:BBa_K823004 J23100], [http://partsregistry.org/Part:BBa_K823005 J23101], [http://partsregistry.org/Part:BBa_K823006 J23102], [http://partsregistry.org/Part:BBa_K823007 J23103], [http://partsregistry.org/Part:BBa_K823008 J23106], [http://partsregistry.org/Part:BBa_K823009 J23107], [http://partsregistry.org/Part:BBa_K823010 J23113], [http://partsregistry.org/Part:BBa_K823011 J23114], [http://partsregistry.org/Part:BBa_K823012 J23115], [http://partsregistry.org/Part:BBa_K823013 J23117], [http://partsregistry.org/Part:BBa_K823014 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. It seems that 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 [http://partsregistry.org/Part:BBa_K823000 P_liaG], [http://partsregistry.org/Part:BBa_K823001 P_liaI], [http://partsregistry.org/Part:BBa_K823003 P_veg] and [http://partsregistry.org/Part:BBa_K823002 P_lepA] are now in the vector pSB1C3 as per the BioBrick standard for the registry.

The Anderson promoters [http://partsregistry.org/Part:BBa_K823004 J23100], [http://partsregistry.org/Part:BBa_K823005 J23101], [http://partsregistry.org/Part:BBa_K823006 J23102], [http://partsregistry.org/Part:BBa_K823007 J23103], [http://partsregistry.org/Part:BBa_K823008 J23106], [http://partsregistry.org/Part:BBa_K823009 J23107], [http://partsregistry.org/Part:BBa_K823010 J23113], [http://partsregistry.org/Part:BBa_K823011 J23114], [http://partsregistry.org/Part:BBa_K823012 J23115], [http://partsregistry.org/Part:BBa_K823013 J23117], [http://partsregistry.org/Part:BBa_K823014 J23118] are now in the Bacillus reporter vector [http://partsregistry.org/Part:BBa_K823025 pSB_Bs3C-luxABCDE] for measuring their activity as luminescence.

The Bacillus promoters [http://partsregistry.org/Part:BBa_K823000 P_liaG], [http://partsregistry.org/Part:BBa_K823001 P_liaI] and [http://partsregistry.org/Part:BBa_K823002 P_lepA] are in the Bacillus reporter vector [http://partsregistry.org/Part:BBa_K823025 pSB_Bs3C-luxABCDE].

14-18 May 2012

Combining the BioBricks and integrating them into different vectors works well! 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 [http://partsregistry.org/Part:BBa_K823000 P_liaG], [http://partsregistry.org/Part:BBa_K823001 P_liaI], [http://partsregistry.org/Part:BBa_K823003 P_veg] and in vector [http://partsregistry.org/Part:BBa_K823021 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 [http://partsregistry.org/Part:BBa_K823021 pSB_Bs1C-lacZ] was finished.

9-13 April 2012

For the knockouts, 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K823026 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 [http://www.ncbi.nlm.nih.gov/pubmed/19554258 J. Kim and W. Schumann (2009)], we decided to knock out genes cwlB, gerD, cwlJ, and sleB. From the research of [http://www.ncbi.nlm.nih.gov/pubmed/11466293 B. Setlow et al (2001)], we also chose cwlD.

5-9 March 2012

We spent two full days (and nights) together planning our project in our "iGEM days." The idea of Beadzillus was born, and we got to know each other better as we divided the modules of the project, and learned each others' strengths and experience in different fields such as microbiology, ecology and biochemistry.

February

20-24 February 2012

Team fully formed!

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