Difference between revisions of "Part:BBa K1044006"

Revision as of 21:43, 27 September 2013

Vinary Vector for E.coli and Agrobacterium

We named BBa_K1044006 "pBI107".This plasmid length is about 9,000 bp.

●Function
File:Biwako-nagahama pBI107 kei1.png File:Bwakonagahama pBI107length kei2.png

pBI107 is improved form of empty backbone vector obtained from pre-existing binary vector of E.coli & Agrobacterium. Selective with Kanamycin, there is possibility of reproduction by both E.coli and Agrobacterium.
pBI107 having both prefix and suffix, we can freely insert the cassette into the cloning site of the BioBrick which gives possibility of different expressions of BioBricks with Agrobacterium.
File:Biwakonagahama pBI107BioBrick kei3.png

Also the possibility of use of OriV can just not be limited with Agrobacterium but also may broaden to be used with other organisms.

●Proof for the reproduction of E.coli’s pBI107 and selection (NPT III)
After cloning the E.coli JM109 with pBI107, we plated the contents to a LB-Agar plate with 50 µg/ml kanamycin and cultured it at 37℃ for overnight. Similar experiment was performed for the E.coli JM109 without cloning to pBI107. From the experiment we found that under 50 µg/ml kanamycin, E.coliJM109 with pBI107 cultured and developed colony but the E.coli without pBI107 did not develop any colony.
From above result it was confirmed that E.coli JM109 cloned with pBI107 can reproduce under the influence of kanamycin. Hence, the functionality of ColE & NPTⅢwith regard to E.coli JM109 was proved.
File:Biwakonagahama pBI107inE.coli kei7.png

●Proof for the reproduction of Agrobacterium’s pBI107 and selection (NPT III)
After cloning Agrobacterium C58 with pBI107, it was plated to LB-Agar plate with 50 µg/ml kanamycin and cultured for 2 days at 30℃. Similar, experiment was carried out for the Agrobacterium C58 without cloning it. Colonies were confirmed for the cloned Agrobacetrium C58 under 50 µg/ml kanamycin but no colonies were found for the normal Agrobacterium C58.
From above result it was confirmed that Agrobacterium C58 cloned with pBI107 can reproduce under the influence of kanamycin. Hence, the functionality of OriV & NPTⅢwith regard to Agrobacterium C58 was proved.
File:Biwakonagahama pBI107inAgroi kei8.png

●Proof for the prefix and suffix of pBI107
We determined the prefix and suffix of the BioBrick cloning site and inserted RFP device(BBa_J04450) into pBI107and cloned E.coli JM109.
Then it was cultured in LB-Agar plate with 50 µg/ml kanamycin and IPTG at 37℃. After 6 hours of culturing, colonies were detected and after 24 hours of culturing the colonies with inserted RFP device were confirmed by the colonies which were shining red.
File:Biwakonagahama pBI107RFPpicture kei4.png

The shining red colonies were extracted and treated with restriction enzyme used for extraction of prefix and suffix. Then the length of vector(pBI107) & insert(RFP device) were confirmed through electrophoresis.
As expected the actual length of pBI107 was 9000bp whereas the extracted RFP device’s length was around 1100bp. File:Biwakonagahama pBI107RFP kei5.png

From the above result, we found out the existence of prefix and suffix as the iGEM standard in pBI107 and hence BioBrick was expressed with E.coli JM109.

Sequence and Features
[Length : about 9,000bp]