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Team:LMU-Munich/Bacillus BioBricks
From 2012.igem.org
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[[Image:LMU-Munich_Introduce_Bacillus.jpg|400px|center]] | [[Image:LMU-Munich_Introduce_Bacillus.jpg|400px|center]] | ||
| - | This <b>B</b>acillus <b>B</b>io<b>B</b>rick <b>B</b>ox contains | + | This <b>B</b>acillus <b>B</b>io<b>B</b>rick <b>B</b>ox contains: |
| + | *'''[[Team:LMU-Munich/Bacillus_BioBricks#Bacillus_vectors|Bacillus vectors]]''' | ||
| + | *Bacillus promoter | ||
| + | *Bacillus reporter | ||
==Bacillus vectors== | ==Bacillus vectors== | ||
Revision as of 17:44, 31 August 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 ]
Bacillus BioBricks
We will create a toolbox of Bacillus BioBricks to contribute to the registry.
We would like to introduce Bacillus to the world of iGEM!!!
This Bacillus BioBrick Box contains:
- Bacillus vectors
- Bacillus promoter
- Bacillus reporter
Bacillus vectors
Here is a list of all the vectors we cloned and used. Please note that pMAD is not in a BioBrick standard, but it is a useful tool to knock out genes inBacillus subtilis.
For the use of our vectors, please see our Protocols page. A general introduction to Bacillus subtilis and its integrative vectors can be found here. All vectors have ampicillin as E. coli resistance and RFP as selection marker.
| name | E. coli res. | B. subt. res. | insertion locus | description | derived from | reference |
| pSBBs1C-lacZ | Amp | Cam | amyE | with lacZ reporter gene | pAC6 | ? |
| pSBBs4S | Amp | Spec | thrC | empty | pDG1731 | ? |
| pSBBs1C | Amp | Cam | amyE | empty | pDG1662 | ? |
| pSBBs4S-PXyl | Amp | Spec | thrC | with Xylose-inducible promoter | pXT | ? |
| pSBBs3C-luxABCDE | Amp | Cam | sacA | with luxABCDE reporter cassette | pAH328 | ? |
| pSBBs0K-Pspac | Amp | Kan | replicative | with IPTG inducible promoter | pDG148 | ? |
| pSBBs2E | Amp | MLS | lacA | empty | pAX01 | ? |
The number in the vector's name codes for the insertion locus and the following letter for the Bacillus subtilis resistance gene according to the following table:
| number | insertion locus | letter | resistance |
| 0 | replicative | C | Chloramphenicol |
| 1 | amyE (amylase) | E | MLS (Erythromycin + Lincomycin) |
| 2 | lacA (laccase ?) | K | Kanamycin |
| 3 | sacA (saccase?) | S | Spectinomycin |
| 4 | thrC (threonine C) |
The concentrations of the antibiotics and the insertion tests can be found in our protocol section.
The promoters we submit:
Pveg, PliaI, PliaG, plepA, Pxyl, Pxyl+XylR
We also tested the Anderson promoter collection in Bacillus subtilis with pSBBs3C-luxABCDE. The data will be online in our data section soon.
Furthermore we plan to submit reporter genes optimized for Bacillus subtilis , namely GFP, lacZ, luc and mKate.
Useful for the registry are also 5 tages in Freiburg Standard (cMyc, 10His, Flag, Strep, HA).
More details to come soon.
Bacillus Promoters
Another goal is to produce promoters of Bacillus subtilis in BioBrick mode and to evaluate them. These well-defined promoters will then be part of the Bacillus BioBrickBox which we will send to the registry but they can also be useful in our project Beadzillus to express our fusion crust proteins on the outside of our spores. Therefore we will use different promoters which are the constitutive promoters from the Anderson collection from the partsregistry, the constitutive promoters PliaG, Pveg and PlepA from B. subtilis as well as the inducible promoter PliaI from B. subtilis. For the characterization of the different promoters we will clone them upstream of reporter genes (lux operon, lacZ) to measure their activity in B. subtilis. Therefore we use e.g. the two reporter vectors from B. subtilis which are also part of the Bacillus BioBrickBox. One of the reporter vectors pSBBs3C-luxABCDE contains the lux operon as a reporter. This is why the activity of the promoter can be measured as luminescence with the plate reader (BioTek) which is a result of the expression of the luciferase. The second reporter vector used for the evaluation of the promoters is pSBBs1C-lacZ which contains the reporter gene lacZ. The promoter activity leads to the production of the enzyme beta-galactosidase which cleaves the substrate ONPG. The product is detectable with the photometer and refers to the promoter activity.
Project Navigation
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Bacillus BioBrickBOX |
SporeCoat FusionProteins |
Germination STOP |
