Difference between revisions of "Part:BBa K382003"
 
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<partinfo>BBa_K382003 short</partinfo>
 
<partinfo>BBa_K382003 short</partinfo>
  
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Vector for transformation of plants. Cloning in E. coli, transfer to Agrobacterium, integrates into plant genome. Kan resistance marker in bacteria, confers Kan resistance in plants. Has the constitutive promoter pENTCUP2 upstream of multiple cloning site for easy expression of genes in plants. Conforms to Silver BioBrick Assembly Standard 21.
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Vector for transformation of plants. Cloning in E. coli, transfer to Agrobacterium, integrates into plant genome. Kan resistance marker in bacteria, confers Kan resistance in plants. Has the constitutive promoter pENTCUP2 upstream of multiple cloning site for easy expression of genes in plants. Conforms to Silver BioBrick Assembly Standard 21.<br>
  
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We used this vector for integration of several gene constructs into ''Arabidopsis''.[http://2010.igem.org/Team:Harvard/results]
 
  
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Plant transformed with this vector:
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'''Confirmed functions''':
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* Kan resistance in bacteria: confirmed by transformation and colony growth of ''E. coli'' (DH5alpha) and ''Agrobacterium tumefaciens''
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* Kan resistance in plants: confirmed by selective germination of transformed Arabidopsis seeds on kanamycin agar plates (See photos [http://2010.igem.org/Team:Harvard/results]).<br><br>
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Confirmation pending: Function of the ENTCUP2 promoter will be tested by checking for expression for several BioBrick parts in adult ''Arabidopsis'' plants. These parts include...
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* LTP knock-down amiRNA and ihpRNA ([[Part:BBa_K382065|BBa_K382065]], [[Part:BBa_K382071|BBa_K382071]])
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* Ger3 knock-down ihpRNA ([[Part:BBa_K382067|BBa_K382067]])
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* BetVI knock-down ihpRNA ([[Part:BBa_K382069|BBa_K382069]])
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* LUT2 knock-down amiRNA ([[Part:BBa_K382061|BBa_K382061]])
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* LYC knock-down amiRNA ([[Part:BBa_K382063|BBa_K382063]])
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* Beta Ohase I knock-down amiRNA ([[Part:BBa_K382062|BBa_K382062]])
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Transformed seeds were plated on 50 micrograms/ml kanamycin in Murashige and Skoog agar and we observed approximately 0.1% transgene expression and sprout survival.
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[[Image:Kanselection.jpg]]<br>
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Successfully transfected kanamycin resistant ''Arabidopsis'' plants, such as the ones shown below, will be tested for BioBrick expression once they have grown large enough for tissue sampling. Shown below are plants transformed with LUT2 [[Part:BBa_K382061|BBa_K382061]] ("C1") and Beta Ohase I knock-down amiRNA [[Part:BBa_K382062|BBa_K382062]] ("C2"):
 
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[[Image:BBa_K382003_plant.jpg|400px]]
 
[[Image:BBa_K382003_plant.jpg|400px]]
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Download the annotated sequence file [http://openwetware.org/images/3/33/V10.gb here]<br><br>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 17:16, 7 November 2010

pORE Expression Series Vector with BioBrick MCS (Kan reistance)

Vector for transformation of plants. Cloning in E. coli, transfer to Agrobacterium, integrates into plant genome. Kan resistance marker in bacteria, confers Kan resistance in plants. Has the constitutive promoter pENTCUP2 upstream of multiple cloning site for easy expression of genes in plants. Conforms to Silver BioBrick Assembly Standard 21.


Confirmed functions:

  • Kan resistance in bacteria: confirmed by transformation and colony growth of E. coli (DH5alpha) and Agrobacterium tumefaciens
  • Kan resistance in plants: confirmed by selective germination of transformed Arabidopsis seeds on kanamycin agar plates (See photos [http://2010.igem.org/Team:Harvard/results]).

Confirmation pending: Function of the ENTCUP2 promoter will be tested by checking for expression for several BioBrick parts in adult Arabidopsis plants. These parts include...

Transformed seeds were plated on 50 micrograms/ml kanamycin in Murashige and Skoog agar and we observed approximately 0.1% transgene expression and sprout survival.

Kanselection.jpg

Successfully transfected kanamycin resistant Arabidopsis plants, such as the ones shown below, will be tested for BioBrick expression once they have grown large enough for tissue sampling. Shown below are plants transformed with LUT2 BBa_K382061 ("C1") and Beta Ohase I knock-down amiRNA BBa_K382062 ("C2"):
BBa K382003 plant.jpg
Download the annotated sequence file [http://openwetware.org/images/3/33/V10.gb here]

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 5269
    Illegal XbaI site found at 5284
    Illegal SpeI site found at 5298
    Illegal PstI site found at 5312
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 5269
    Illegal SpeI site found at 5298
    Illegal PstI site found at 5312
    Illegal NotI site found at 5275
    Illegal NotI site found at 5305
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 5269
    Illegal BglII site found at 5182
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 5269
    Illegal suffix found in sequence at 5298
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 5269
    Illegal XbaI site found at 5284
    Illegal SpeI site found at 5298
    Illegal PstI site found at 5312
    Illegal NgoMIV site found at 625
    Illegal NgoMIV site found at 749
    Illegal NgoMIV site found at 2181
    Illegal NgoMIV site found at 2772
    Illegal NgoMIV site found at 7160
    Illegal NgoMIV site found at 7795
    Illegal AgeI site found at 341
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 7644
    Illegal SapI.rc site found at 7854