Difference between revisions of "Part:BBa K1856000"
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<partinfo>BBa_K1856000 short</partinfo>
 
<partinfo>BBa_K1856000 short</partinfo>
  
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bacA promoter fused to citrine
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bacA promoter assembled to citrine and T7 terminator
  
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===Usage and Biology===
 
===Usage and Biology===
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bacA is an inducible promoter native to rhizobium species (notably R. tropici and S. meliloti) that is induced by flavonoids such as apigenin. The Yale iGEM team has assembled this promoter to citrine (an improved version of YFP, with excitation peak at 514nm and emission peak at 527nm) and a T7-terminator to quantify the level of expression in E. coli and in non-model organism hosts.
  
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This construct has been successfully cloned into E. coli using the broad-host range vector pKT230, a RSF1010 derived plasmid, as well as using the pPZP200 plasmid which can be transformed into agrobacterium and rhizobium. Leaky expression of citrine was observed.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1856000 SequenceAndFeatures</partinfo>
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RSF1010 plasmids belong to the IncQ group and can be transformed into a wide range of gram-negative bacteria as well as some gram-positive bacteria. These include cyanobacteria genera such as Synechocystis, Synechococcus and Anabaena, rhizobium genera such as Rhizobium and Sinorhizobium, and other genera such as Pseudomonas, Streptomyces and Mycobacterium. The plasmid is mobilizable by conjugation.
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We have used a derivative of pKT230 that is compatible with ligation-independent cloning using BsaI. pKT230 has a kanamycin-resistance marker.
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pPZP200 is a agrobacterium binary plasmid that can also be transformed into rhizobium genera. We also used a derivative that is LIC compatible for high-efficiency cloning of our constructs. pPZP200 has a spectinomycin-resistance marker.
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Beyond the E. coli work, the construct has also been successfully cloned into S. meliloti and PCR-verified.
  
 
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==Description==
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==Characterization==
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Members of the Anderson promoter collection are suitable for general protein expression in ''E. coli'' and likely other prokaryotes.  The collection is known to cover a range of activities so by testing a few promoters it should be possible to find a promoter activity that suits your application.  The promoters were recovered from a library screen by [https://andersonlab.qb3.berkeley.edu/ Chris Anderson].
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[[Image:Expression Level for bacA-citrine (pKT230-Lic).jpg|300px|right]]
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===Measured strength===
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Leaky expression of BacA was observed, with 3 times more fluorescence than background readings.
  
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Parts J23100 through J23119 are a family of constitutive promoter parts isolated from a small combinatorial library. J23119 is the "consensus" promoter sequence and the strongest member of the family. The NheI and AvrII restriction sites present within these promoter parts make them a scaffold for further modification.
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These fluorescence readings are part of a series of readings taken using different inducible and constitutive promoters to drive citrine expression. See parts [https://parts.igem.org/Part:BBa_K1856001 K1856001], [https://parts.igem.org/Part:BBa_K1856002 K1856002], [https://parts.igem.org/Part:BBa_K1856003 K1856003], [https://parts.igem.org/Part:BBa_K1856004 K1856004], [https://parts.igem.org/Part:BBa_K1856005 K1856005], [https://parts.igem.org/Part:BBa_K1856006 K1856006] for details of the full characterization set.
  
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==Characterization==
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==Obtaining the BacA promoter-citrine construct==
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[[Image:BerkiGEM2006-PromotersEppendorfs.jpg|300px|right]]
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The sequences of construct can be found via the table below.  The physical DNA can be obtained from:
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===Measured strengths===
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Reported activities of the promoters are given as the relative fluorescence of these plasmids in strain TG1 grown in LB media to saturation.  See part [https://parts.igem.org/Part:BBa_J61002 J61002] for details on their use.
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==Obtaining the Anderson promoter collection==
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'''Via request''': The Yale iGEM team has the construct cloned into the broad-host range plasmid, pKT230, a RSF1010 derivative. This plasmid is known to work in cyanobacteria genera such as Synechocystis, Synechococcus and Anabaena, in rhizobium genera such as Rhizobium and Sinorhizobium. The team has also cloned the construct into pPZP200. Both are extremely versatile expression vectors. Please contact the Yale team if you would like to request for an aliquot of the constructs in these plasmids.
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[[Image:Expression_Level_for_melA-citrine_%28pKT230-Lic%29.jpg|right|150px|right]]
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The sequences of the Anderson promoters can be found via the table below.  To obtain the physical DNA, we recommend two approaches - <br>
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'''Via ''de novo'' synthesis''': Since the promoters are short sequences, they can be easily and cheaply ordered as two single-stranded complementary oligo's and annealed. See [[Help:Promoters/Construction|here]] for a tutorial on how to construct short parts via oligo annealing.
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'''Via the Registry distribution''': The promoters are included in the Registry distribution.  All parts except <partinfo>J23119</partinfo> are present in plasmid <partinfo>J61002</partinfo>. This places the RFP downstream of the promoter. Part <partinfo>J23119</partinfo> is present in <partinfo>pSB1A2</partinfo>.
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'''Via the Registry distribution''': The constructs are included in the Registry distribution, cloned in [https://parts.igem.org/Part:pSB1C3 pSB1C3] backbone.
 
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1856000 SequenceAndFeatures</partinfo>

Latest revision as of 22:59, 20 September 2015

bacA-citrine-T7 terminator

bacA promoter assembled to citrine and T7 terminator

Usage and Biology

bacA is an inducible promoter native to rhizobium species (notably R. tropici and S. meliloti) that is induced by flavonoids such as apigenin. The Yale iGEM team has assembled this promoter to citrine (an improved version of YFP, with excitation peak at 514nm and emission peak at 527nm) and a T7-terminator to quantify the level of expression in E. coli and in non-model organism hosts.

This construct has been successfully cloned into E. coli using the broad-host range vector pKT230, a RSF1010 derived plasmid, as well as using the pPZP200 plasmid which can be transformed into agrobacterium and rhizobium. Leaky expression of citrine was observed.

RSF1010 plasmids belong to the IncQ group and can be transformed into a wide range of gram-negative bacteria as well as some gram-positive bacteria. These include cyanobacteria genera such as Synechocystis, Synechococcus and Anabaena, rhizobium genera such as Rhizobium and Sinorhizobium, and other genera such as Pseudomonas, Streptomyces and Mycobacterium. The plasmid is mobilizable by conjugation.

We have used a derivative of pKT230 that is compatible with ligation-independent cloning using BsaI. pKT230 has a kanamycin-resistance marker.

pPZP200 is a agrobacterium binary plasmid that can also be transformed into rhizobium genera. We also used a derivative that is LIC compatible for high-efficiency cloning of our constructs. pPZP200 has a spectinomycin-resistance marker.

Beyond the E. coli work, the construct has also been successfully cloned into S. meliloti and PCR-verified.


Characterization

Expression Level for bacA-citrine (pKT230-Lic).jpg

Measured strength

Leaky expression of BacA was observed, with 3 times more fluorescence than background readings.

These fluorescence readings are part of a series of readings taken using different inducible and constitutive promoters to drive citrine expression. See parts K1856001, K1856002, K1856003, K1856004, K1856005, K1856006 for details of the full characterization set.

Obtaining the BacA promoter-citrine construct

The sequences of construct can be found via the table below. The physical DNA can be obtained from:

Via request: The Yale iGEM team has the construct cloned into the broad-host range plasmid, pKT230, a RSF1010 derivative. This plasmid is known to work in cyanobacteria genera such as Synechocystis, Synechococcus and Anabaena, in rhizobium genera such as Rhizobium and Sinorhizobium. The team has also cloned the construct into pPZP200. Both are extremely versatile expression vectors. Please contact the Yale team if you would like to request for an aliquot of the constructs in these plasmids.

Via the Registry distribution: The constructs are included in the Registry distribution, cloned in pSB1C3 backbone.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 920
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 920
    Illegal NotI site found at 945
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 920
    Illegal BamHI site found at 914
    Illegal XhoI site found at 954
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 920
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 920
  • 1000
    COMPATIBLE WITH RFC[1000]