Team:LMU-Munich/Bacillus BioBricks

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Revision as of 17:42, 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 compatible vectors and useful promoters.

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
pSB_Bs1C-lacZ	Amp	Cam	amyE	with lacZ reporter gene	pAC6	?
pSB_Bs4S	Amp	Spec	thrC	empty	pDG1731	?
pSB_Bs1C	Amp	Cam	amyE	empty	pDG1662	?
pSB_Bs4S-P_Xyl	Amp	Spec	thrC	with Xylose-inducible promoter	pXT	?
pSB_Bs3C-luxABCDE	Amp	Cam	sacA	with luxABCDE reporter cassette	pAH328	?
pSB_Bs0K-P_spac	Amp	Kan	replicative	with IPTG inducible promoter	pDG148	?
pSB_Bs2E	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 pSB_Bs3C-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 P_liaG, P_veg and P_lepA from B. subtilis as well as the inducible promoter P_liaI 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 pSB_Bs3C-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 pSB_Bs1C-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

+--+--+--+--+--+--+--	+--+--+--+--+--	+--+--+--+--+--+--	+--+--+--+--+--+--
Bacillus Intro	Bacillus BioBrickBOX	SporeCoat FusionProteins	Germination STOP

Retrieved from "http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks"

@@ Line 13: / Line 13: @@
 This <b>B</b>acillus <b>B</b>io<b>B</b>rick <b>B</b>ox contains compatible vectors and useful promoters.
+==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.
@@ Line 136: / Line 138: @@
 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 '''Bead'''zillus 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 P<sub>''liaG''</sub>, P<sub>''veg''</sub> and P<sub>''lepA''</sub> from ''B. subtilis'' as well as the inducible promoter P<sub>''liaI''</sub> 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 pSB<sub>Bs</sub>3C-<i>luxABCDE</i> 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 pSB<sub>Bs</sub>1C-''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.