Difference between revisions of "Part:BBa K382003"

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...

LTP knock-down amiRNA and ihpRNA (BBa_K382065, BBa_K382071)
Ger3 knock-down ihpRNA (BBa_K382067)
BetVI knock-down ihpRNA (BBa_K382069)
LUT2 knock-down amiRNA (BBa_K382061)
LYC knock-down amiRNA (BBa_K382063)
Beta Ohase I knock-down amiRNA (BBa_K382062)

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.

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"):

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

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K382003 short</partinfo>
-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 23.<br>
+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>
@@ Line 25: / Line 25: @@
 <br>
 [[Image:BBa_K382003_plant.jpg|400px]]
+<br>
+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