Difference between revisions of "Part:BBa K2995000"

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Constitutive promoter for expression in E. coli and B. japonicum.
 
Constitutive promoter for expression in E. coli and B. japonicum.
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(Waterloo iGEM 2019)
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For the silver BioBrick criteria, we decided to BioBrick and characterized the promoter Paph (also referred to as P-nptII) from the plasmid pRJPaph-bjGFP (https://www.addgene.org/67032/). This promoter was used in our project this year to express our pesticide resistance pathways along with GFP in B.japonicum. We thought this would be a valuable addition to the registry since few parts have confirmed function in organisms like B. japonicum. We recommend that teams use this promoter when constructing parts to be used in B. japonicum however, expression also works in e.coli.
  
 
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(Waterloo iGEM 2019)  
 
(Waterloo iGEM 2019)  
  
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To characterize our promoter we cloned it upstream of the RBS in BBa_E0240 (RBS-GFP-Term) and compared it's GFP expression with the already well characterized BBa_J23101 promoter in BBa_I20260. To limit the impact of other variables we insured that BBa_J23101 and BBa_E0240 shared the same backbone, RBS and terminator. In addition to characterizing this promoter with respect to BBa_I20260 we also confirmed proper function of this promoter in the pRJPaph-bjGFP plasmid which was used for expression in Bradyrhizobium for our 2019 project. 
 
  
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Note: empty DH5alpha was used as a negative control for GFP expression
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To characterize our promoter we cloned it upstream of the RBS in <partinfo>BBa_E0240</partinfo> (RBS-GFP-Term) and compared it's GFP expression with the already well characterized <partinfo>BBa_J23101</partinfo> promoter in <partinfo>BBa_I20260</partinfo>. To limit the impact of other variables we insured that <partinfo>BBa_I20260</partinfo> and <partinfo>BBa_E0240</partinfo> shared the same backbone, RBS and terminator. In addition to characterizing this promoter with respect to<partinfo>BBa_I20260</partinfo> we also confirmed proper function of this promoter in the pRJPaph-bjGFP plasmid which was used for expression in Bradyrhizobium for our 2019 project. 
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<strong>Note:</strong><br> empty DH5alpha was used as a negative control for GFP expression
  
 
1. Cell were grown overnight in LB media at 37 degrees
 
1. Cell were grown overnight in LB media at 37 degrees
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2. The following morning GFP expression was quantified using the Synergy 4 BioTek plate reader.
 
2. The following morning GFP expression was quantified using the Synergy 4 BioTek plate reader.
  
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The graph below indicates the results from this characterization experiment:
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<strong>The graph below indicates the results from this characterization experiment:</strong><br>
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[[File:T--waterloo--silvergraph.jpg|480px|thumb|left|Figure 1: OD-corrected GFP fluorescence for characterization of P-nptII (Excitation: 485, Emmission: 525)]]
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Both plasmids containing the promoter P-nptII (pRJPaph-bjGFP and BBa_E0240+BBa_K2995000) show a significant difference in fluorescence vs the empty (DH5alpha) indicating that our promoter is functional. In addition, this data shows that the promoter P-nptII is weaker in comparison with the BBa_J23101 and should be used when weaker expression is desired.

Latest revision as of 00:58, 22 October 2019


Paph promoter

Constitutive promoter for expression in E. coli and B. japonicum.

(Waterloo iGEM 2019)

For the silver BioBrick criteria, we decided to BioBrick and characterized the promoter Paph (also referred to as P-nptII) from the plasmid pRJPaph-bjGFP (https://www.addgene.org/67032/). This promoter was used in our project this year to express our pesticide resistance pathways along with GFP in B.japonicum. We thought this would be a valuable addition to the registry since few parts have confirmed function in organisms like B. japonicum. We recommend that teams use this promoter when constructing parts to be used in B. japonicum however, expression also works in e.coli.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

(Waterloo iGEM 2019)


To characterize our promoter we cloned it upstream of the RBS in BBa_E0240 (RBS-GFP-Term) and compared it's GFP expression with the already well characterized BBa_J23101 promoter in BBa_I20260. To limit the impact of other variables we insured that BBa_I20260 and BBa_E0240 shared the same backbone, RBS and terminator. In addition to characterizing this promoter with respect toBBa_I20260 we also confirmed proper function of this promoter in the pRJPaph-bjGFP plasmid which was used for expression in Bradyrhizobium for our 2019 project.

Note:
empty DH5alpha was used as a negative control for GFP expression

1. Cell were grown overnight in LB media at 37 degrees

2. The following morning GFP expression was quantified using the Synergy 4 BioTek plate reader.

The graph below indicates the results from this characterization experiment:

Figure 1: OD-corrected GFP fluorescence for characterization of P-nptII (Excitation: 485, Emmission: 525)



















Both plasmids containing the promoter P-nptII (pRJPaph-bjGFP and BBa_E0240+BBa_K2995000) show a significant difference in fluorescence vs the empty (DH5alpha) indicating that our promoter is functional. In addition, this data shows that the promoter P-nptII is weaker in comparison with the BBa_J23101 and should be used when weaker expression is desired.