Team:LMU-Munich/Project

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=== Evaluate Promoters ===
=== Evaluate Promoters ===
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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 one project 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 ''Bacillus 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 luceiferase. 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.
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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 one project 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<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 luceiferase. 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.
There are three types of promoters which will be evaluated in this project as a part of the so-called BioBrickBox:
There are three types of promoters which will be evaluated in this project as a part of the so-called BioBrickBox:
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The first group of promoters are the promoters of the Anderson collection which we call ''Anderson promoters''. They have already been measured in ''Escherichia coli'' and they all showed a constitutive behaviour but with a different strength. In this project these Anderson promoters were measured in ''B. subtilis''. As they have already been measured in ''E. coli'', they are already in the partsregistry in the vector where they are fused to the so-called ''Red fluorescent protein'' (RFP). For evaluation they are cut out of the vector and then cloned in the expression vector pSB<sub>Bs</sub>3C-<i>luxABCDE</i> from the BioBrickBox containing the ''lux'' operon as a reporter for promoter activity. The gene expression which correlates to the promoter activity leads to the expression of the lux operon with the luciferase. The luminescence which is produced by the luciferase can be measured with the plate reader (BioTek).
+
The first group of promoters are the promoters of the Anderson collection which we call ''Anderson promoters''. They have already been measured in ''Escherichia coli'' and they all showed a constitutive behaviour but with a different strength. In this project these Anderson promoters will be measured in ''B. subtilis''. As they have already been evaluated in ''E. coli'', they are already in the partsregistry in the vector where they are fused to the so-called ''Red fluorescent protein'' (RFP). For evaluation they are cut out of the vector and then cloned in the expression vector pSB<sub>Bs</sub>3C-<i>luxABCDE</i> from the BioBrickBox containing the ''lux'' operon as a reporter for promoter activity. The gene expression which correlates to the promoter activity leads to the expression of the ''lux'' operon with the luciferase. The luminescence which is produced by the luciferase can be measured with the plate reader (BioTek).
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The second group of promoters are constitutive promoters from B. subtilis. We will evaluate the promoters PliaG, Pveg and PlepA. Therefore we will use both reporter vectors pSB<sub>Bs</sub>3C-<i>luxABCDE</i> and pSB<sub>Bs</sub>1C-''lacZ'' to evaluate the promoters with different reporters, the ''lux'' operon and the ''lacZ'' gene. Therefore the promoters will be amplified from the genom of B. subtilis with primers that conatin the restriction sites of the BioBrick standard. Then these constitutive promoters will be cloned into the empty vector pSB1C3 to send them to the registry as well as the two reporter vectors to evaluate their strength in ''B. subtilis''.
+
The second group of promoters are constitutive promoters from ''B. subtilis''. We will evaluate the promoters P<sub>''liaG''</sub>, P<sub>''veg''</sub> and P<sub>''lepA''</sub>. Therefore we will use the reporter vectors pSB<sub>Bs</sub>3C-<i>luxABCDE</i> and pSB<sub>Bs</sub>1C-''lacZ'' to evaluate the promoters with different reporters, the ''lux'' operon and the ''lacZ'' gene. Therefore the promoters will be amplified from the genom of ''B. subtilis'' with primers that conatin the restriction sites of the BioBrick standard. Then these constitutive promoters will be cloned into the empty vector pSB1C3 to send them to the registry as well as the two reporter vectors to evaluate their strength in ''B. subtilis''.
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The last group of promoters consist of inducible promoters of ''B. subtilis'' e.g. P''<sub>liaI</sub>''. They are useful if you want to turn the system on in a certain moment because these promoters need an inducer to start transcription. P''<sub>liaI</sub>' can be induced by antibiotics which interact with the lipidII cycle, e.g. bacitracin. In this project this promoter is also evaltuated with the reporter vector pSB<sub>Bs</sub>3C-<i>luxABCDE</i> which contains the lux operon as a reporter and with the vector pSB<sub>Bs</sub>1C-''lacZ'' which conatins the ''lacZ'' reporter gene. Therefore the promoters will be amplified from the genom of B. subtilis with primers that conatin the restriction sites of the BioBrick standard. Then these constitutive promoters will be cloned into the empty vector pSB1C3 to send them to the registry as well as the two reporter vectors to evaluate their strength in ''B. subtilis''.
+
The last group of promoters consists of inducible promoters of ''B. subtilis'' e.g. P''<sub>liaI</sub>''. They are useful to decide when to turn on gene expression because these promoters need an inducer to start transcription. P''<sub>liaI</sub>' can be induced by antibiotics which interact with the lipidII cycle, e.g. bacitracin. In this project this promoter is evaltuated like the constitutive promoters with the reporter vector pSB<sub>Bs</sub>3C-<i>luxABCDE</i> which contains the lux operon as a reporter and with the vector pSB<sub>Bs</sub>1C-''lacZ'' which conatins the ''lacZ'' reporter gene. Therefore the promoters will be amplified from the genom of ''B. subtilis'' with primers that conatin the restriction sites of the BioBrick standard. Then these inducible promoters will be cloned into the empty vector pSB1C3 to send them to the registry as well as the two reporter vectors to evaluate their strength in ''B. subtilis''. To turn the promoter on we have to add an inducer.

Revision as of 20:26, 18 August 2012

iGEM Ludwig-Maximilians-Universität München Beadzillus

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