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Team:LMU-Munich/Weekly Journal
From 2012.igem.org
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<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html> | <img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html> | ||
PspoIVB-ECF41 in pSB<sub>BS</sub>4S, PydfG/PsspK/PspoIVB in pSB<sub>BS</sub>3C-''luxABCDE'' brought into ''B. subtilis''</p> | PspoIVB-ECF41 in pSB<sub>BS</sub>4S, PydfG/PsspK/PspoIVB in pSB<sub>BS</sub>3C-''luxABCDE'' brought into ''B. subtilis''</p> | ||
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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<sub>BS</sub>1C! Hopefully it will integrate easily! :D | 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<sub>BS</sub>1C! Hopefully it will integrate easily! :D | ||
Now the bad one: Finally we could start with the P<sub>cgeA</sub> evaluation! But contrary to our expectations this promoter did not show any activity :( </p> | Now the bad one: Finally we could start with the P<sub>cgeA</sub> evaluation! But contrary to our expectations this promoter did not show any activity :( </p> | ||
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<img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html> | <img src="https://static.igem.org/mediawiki/2012/c/c1/SporeCoat.png" height=40"/></a></html> | ||
At last Bacillus was keen on integrating the pSB<sub>BS</sub>3C-luxABCDE-P<sub>''cgeA''</sub>!! And the clean deletions of CotZ and CgeA worked out, too!</p> | At last Bacillus was keen on integrating the pSB<sub>BS</sub>3C-luxABCDE-P<sub>''cgeA''</sub>!! And the clean deletions of CotZ and CgeA worked out, too!</p> | ||
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<img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html> | <img src="https://static.igem.org/mediawiki/2012/7/78/BacillusBioBrickBox.png" height=40"/></a></html> | ||
Plate reader measurements of the ''Bacillus'' promoters [http://partsregistry.org/Part:BBa_K823000 P<sub>''liaG''</sub>], [http://partsregistry.org/Part:BBa_K823001 P<sub>''liaI''</sub>] and [http://partsregistry.org/Part:BBa_K823002 P<sub>''lepA''</sub>] finished. Look at [https://2012.igem.org/Team:LMU-Munich/Data Data]!</p> | Plate reader measurements of the ''Bacillus'' promoters [http://partsregistry.org/Part:BBa_K823000 P<sub>''liaG''</sub>], [http://partsregistry.org/Part:BBa_K823001 P<sub>''liaI''</sub>] and [http://partsregistry.org/Part:BBa_K823002 P<sub>''lepA''</sub>] finished. Look at [https://2012.igem.org/Team:LMU-Munich/Data Data]!</p> | ||
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'''16-20 July 2012''' | '''16-20 July 2012''' | ||
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<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html> | <img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html> | ||
| - | + | We created the single mutant ''cwlD''::kan.</p> | |
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<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html> | <img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html> | ||
| - | + | We successfully created our first single knockouts of germination genes using a resistance cassettes: ''sleB''::mls, ''gerD''::cm and ''cwlJ''::spec. | |
Jara tried knocking out ''cwlB'' using the kan resistance cassette. Mutants of ''cwlB''::kan grew very poorly.</p> | Jara tried knocking out ''cwlB'' using the kan resistance cassette. Mutants of ''cwlB''::kan grew very poorly.</p> | ||
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<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html> | <img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html> | ||
| - | + | We tried knocking out ''cwlB'' using the tet resistance cassette. Mutants of ''cwlB''::tet grew very poorly.</p> | |
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<img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html> | <img src="https://static.igem.org/mediawiki/2012/f/f6/GerminationSTOP.png" height=40"/></a></html> | ||
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''.</p> | 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''.</p> | ||
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'''20-24 February 2012''' | '''20-24 February 2012''' | ||
Revision as of 14:36, 25 September 2012
The LMU-Munich team is exuberantly happy about the great success at the World Championship Jamboree in Boston. Our project Beadzillus finished 4th and won the prize for the "Best Wiki" (with Slovenia) and "Best New Application Project".
[ more news ]
Weekly Journal
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September
24-28 September 2012
17-21 September 2012
Finished cloning of mKate2 constructs. Now the evaluation of this reporter BioBrick with three different promoters, PliaI, PlepA and the Anderson promoter J23101, can start.
Finished cloning of Sporo vector in pSB1C3. Now, this device will be cloned into pSBBs4S.
Inverter measured quantitavely via ONPG assay
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 significant 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!
PspoIVB-ECF41 in pSBBS4S, PydfG/PsspK/PspoIVB in pSBBS3C-luxABCDE brought into B. subtilis
3-7 September 2012
All our CotZ-GFP variants were examined with fluorescence microscopy! The brightest spores derived from our Pcotyz-cotZrep-gfp-terminator mutants!
Plates of our spores diluted at 10-2, 10-4 and 10-6 from the germination assay 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.
PsspK, PspoIVB and PydfG cloned into pSB1C3 and verified by sequencing
PspoIVB-ECF41 cloned into pSBBS4S and verified by sequencing
PydfG/PsspK/PspoIVB brought into pSBBS3C-luxABCDE
lacZ was succesfully cloned into [http://partsregistry.org/Part:BBa_K823024 pSBBs4S-PXyl 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.
Jara 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.
Germination assay was performed on triple and quadruple mutants.
MazF and ECF41 cloned into pSB1C3 and verified by sequencing
The BioBrick lacZ for B. subtilis was shown to be functional in pSBBs0K-Pspac 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 (Julia).
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 pSBBS1C! Hopefully it will integrate easily! :D
Now the bad one: Finally we could start with the PcgeA evaluation! But contrary to our expectations this promoter did not show any activity :(
13-17 August 2012
Jara and Jenny 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.
ß-glactosiase assay of the Anderson promoters J23100, J23102, J23103 in pSBBs1C-lacZ in B. subtilis.
The xylose-inducible promoter with the according repressor (which has a constitutive promoter, RBS and terminator) PXyl + XylR was cloned into pSB1C3 and sequenced.
At last Bacillus was keen on integrating the pSBBS3C-luxABCDE-PcgeA!! And the clean deletions of CotZ and CgeA worked out, too!
6-10 August 2012
Jara and Jenny 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. We also created the resistance cassette knockout cwlB::kan.
Finally, the last PstI site could be removed and pSBBs3C-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 Practise week!
Form 23rd to 25th 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 PliaG, PliaI and PlepA 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 pSBBs3C-luxABCDE completed. Look at Data Anderson promoters!
Cloning of the Anderson promoters J23100, J23102, J23103, J23106 in the reporter vector pSBBs1C-lacZ for β-galactosidase assays finished.
9-13 July 2012
The vectors pSBBs4S-PXyl , pSBBs1C-lacZ </b> and pSBBs4S 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 protocoll length... What took even longer is the pSBBS3C-luxABCDE-PcgeA, but it is finished now!
2-6 July 2012
Clean deletions of germination genes sleB and cwlB from PCR accomplished. DNA purified and frozen to be later transformed with Bacillus.
β-galactosidase assays of the promoters PliaG, PliaI, Pveg 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 Pcotyz and Pcotv are running! Like expected they both show activity in the late stationary phase, thus during sporulation!
June
25-29 June 2012
pSBBS3C-luxABCDE-Pcotyz and pSBBS3C-luxABCDE-Pcotv 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
Clean deletions of germination genes cwlD, cwlJ, and gerD from PCR accomplished. DNA purified and frozen to be later transformed with Bacillus.
4-8 June 2012
Still working on the Promoter-Gene and now GFP-Terminator constructs!
May
28-1 June 2012
GFP in BioBrick standard is readyyyy!
21-25 May 2012
pSBBS3C-luxABCDE-Pcotyz and pSBBS3C-luxABCDE-Pcotv are ready for transformation into Bacillus!
Promoters PliaG, PliaI, Pveg and PlepA 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 pSBBs3C-luxABCDE for measuring their activity as luminescence.
The Bacillus promoters PliaG, PliaI and PlepA are in the Bacillus reporter vector pSBBs3C-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 PliaG, PliaI, Pveg and in vector pSBBs1C-lacZ finished.
April
30 April-4 Mai 2012
We have the first essential BioBricks for this module: pSB1C3-CotZ, pSB1C3-PcotV, pSB1C3-PcotYZ, pSB1C3-PcgeA, pSB1C3-CgeA! Still working on the pSB1C3-GFP.
23-27 April 2012
pSBBs3C-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 pSBBs1C-lacZ </b> was finished.
9-13 April 2012
We created the single mutant cwlD::kan.
2-6 April 2012
We successfully created our first single knockouts of germination genes using a resistance cassettes: sleB::mls, gerD::cm and cwlJ::spec.
Jara tried knocking out cwlB using the kan resistance cassette. Mutants of cwlB::kan grew very poorly.
With pSBBs0K-Pspac our first vector for our Bacillus BioBrick Box was completed.
March
26-30 March 2012
We tried knocking out cwlB using the tet resistance cassette. Mutants of cwlB::tet grew very poorly.
19-23 March 2012
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!
Antibiotic abbreviation legend:
cm: chloramphenicol
kan: kanamycin
mls: Macrolide-Lincosamide-Streptogramin B
spec: spectinomycin
tet: tetracycline
