Difference between revisions of "Part:BBa K3866000"

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    [[Image:T--Thessaly--aracsnap.png|900px|thumb|none|<I><b>Figure 1.</b> The level 0 module : pupd2- araC:araBAD (illustration from SnapGene)</i>]]
  
  
 
===Usage and Biology===
 
===Usage and Biology===
 
The PBAD promoter, derived from the arabinose operon andis an inducible promoter for arabinose.
 
The PBAD promoter, derived from the arabinose operon andis an inducible promoter for arabinose.
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The use of an inducible promoter provides a most reversible and flexible gene circuit and at the same time exhibits a higher efficiency and lower side effects as cell death.(Kallunki, Barisic, Jäättelä and Liu, 2019). The araC gene, is a regulatory gene and is located upstream of the L-arabinose operon, encodes a positive regulatory protein AraC, required for L-arabinose utilization in Escherichia coli. The araC gene has a constitutive promoter.
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The use of an inducible promoter provides a most reversible and flexible gene circuit and at the same time exhibits a higher efficiency and lower side effects as cell death.(Kallunki, Barisic, Jäättelä and Liu, 2019). The araC gene, is a regulatory gene and is located upstream of the L-arabinose operon, encodes a positive regulatory protein AraC, required for L-arabinose utilization in Escherichia coli. The promoter is highly inhibited from Glucose. The araC gene has a constitutive promoter.
  
 
===Design Notes===
 
===Design Notes===
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<p style="text-align: center;">
 
<p style="text-align: center;">
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     [[Image:T--Thessaly--GB-GGAG-AATG.jpeg |900px|thumb|none|<i><b>Figure 1.</b>The overhangs of this part in the GoldenBraid Grammar.</i>]]
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     [[Image:T--Thessaly--GB-GGAG-AATG.jpeg |900px|thumb|none|<i><b>Figure 2.</b>The overhangs of this part in the GoldenBraid Grammar.</i>]]
  
 
===Verification of Cloning===
 
===Verification of Cloning===
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[[File:T--Thessaly--arac.png|700px|thumb|none|<i><b>Fig.2:</b>(U=Uncut C=Cut) Restriction Digestion of araC:ParaBAD with EcoRI+ BamHI. Positive clones 1, 2 Expected bands 2160, 1184</i>]]
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[[File:T--Thessaly--arac.png|700px|thumb|none|<i><b>Fig.3:</b>(U=Uncut C=Cut) Restriction Digestion of araC:ParaBAD with EcoRI+ BamHI. Positive clones 1, 2 Expected bands 2160, 1184</i>]]
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===Experimental Use and Experience===
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This part showed fuctionality at the following parts. <bbpart>BBa_K3866009</bbpart>  <bbpart>BBa_K3866010</bbpart> <bbpart>BBa_K3866026</bbpart> <bbpart>BBa_K3866027</bbpart> <bbpart>BBa_K3866028</bbpart>
  
 
===Source===
 
===Source===

Latest revision as of 22:34, 25 September 2021


araC-ParaBAD:RBS GB compatible with A1-B2


Figure 1. The level 0 module : pupd2- araC:araBAD (illustration from SnapGene)


Usage and Biology

The PBAD promoter, derived from the arabinose operon andis an inducible promoter for arabinose. The use of an inducible promoter provides a most reversible and flexible gene circuit and at the same time exhibits a higher efficiency and lower side effects as cell death.(Kallunki, Barisic, Jäättelä and Liu, 2019). The araC gene, is a regulatory gene and is located upstream of the L-arabinose operon, encodes a positive regulatory protein AraC, required for L-arabinose utilization in Escherichia coli. The promoter is highly inhibited from Glucose. The araC gene has a constitutive promoter.

Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is present in pUPD2 BBa_K3505007 and has overhangs compatible for GoldenBraid cloning. The CDS has position A1-B2

Figure 2.The overhangs of this part in the GoldenBraid Grammar.

Verification of Cloning

Fig.3:(U=Uncut C=Cut) Restriction Digestion of araC:ParaBAD with EcoRI+ BamHI. Positive clones 1, 2 Expected bands 2160, 1184

Experimental Use and Experience

This part showed fuctionality at the following parts. BBa_K3866009 BBa_K3866010 BBa_K3866026 BBa_K3866027 BBa_K3866028

Source

From the iGEM Distribution Kit 2019.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1148
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 983
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 965


References

  • Kallunki, Barisic, Jäättelä and Liu, 2019. How to Choose the Right Inducible Gene ExpressionSystem for Mammalian Studies?Cells, 8(8), p.796.