Difference between revisions of "Part:BBa K274003:Experience"

Revision as of 22:49, 3 October 2012

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Applications of BBa_K274003

User Reviews

E. coli turned dark green after transformation of this plasmid. This is unexpected as there is no promoter upstream of the operon.

BBa_K274003 Review No.2 UCL_London, Xiang Chen

Diagnostic DNA gel was run to test all 4 restriction sites. BBa_K274003 was cut with XbaI, PstI, EcoRI & SpeI, XbaI & PstI. A gel picture is shown as below. This plasmid did not cut with Xba I restriction site in our hands. As such we could not use it to assemble after other parts.

Characterization by 2012 UCSF iGEM Team

We also attempted to use this part as a PCR template and were unsuccessful. We obtained a plasmid containing these enzymes from another source (Dueber Lab, UC Berkeley) and have re-deposited this part (in a new form) with the registry as
- Part: BBa_K726016

The purpose of this part is to produce prodeoxyviolaceinic acid, a green pigment. However, only the genes for producing the enzymes VioA, VioB, and VioE are necessary to produce the green pigment. Therefore, we have deposited our part (lacking VioD) as a smaller and improved version of BBa K274003. Additionally, BBa_K726016 has a T7 promoter and we have shown that it is able to make the green pigment:

Characterisation by Wits South Africa

We attempted to try and use the Dark Green E.chromi biobrick (BBa_K274003) in our machine constructs. Although the part was excised using EcoR1 and Spe1 (Fermentas), when we attempted to clone anything in front of this part, by digestion with EcoR1 and Xba1 (Fermentas), we could not obtain any positive clones.

Suspecting that perhaps the restriction sites were not present, we tested the plasmid samples through restriction digests.

Figure 1. E.chromi Dark Green digested with various restriction enzymes to confirm the presence of the Biobrick restriction sites.

As can be noted from Figure 1, the Xba1 site is not present in the Dark Green E.chromi Biobrick part, although the Spe1 and EcoR1 sites are present.

UNIQ17ccdf3e87ea363b-partinfo-00000001-QINU UNIQ17ccdf3e87ea363b-partinfo-00000002-QINU