Team:Yale/Our Project/Methods

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(1) phsABC gene and vector
(1) phsABC gene and vector
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(4) Terminator
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<a id="link" href="javascript:ReverseDisplay('background')">Read more about the background of the PHS gene</a>
<a id="link" href="javascript:ReverseDisplay('background')">Read more about the background of the PHS gene</a>
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</li> PHS gene: Important part of anaerobic bacterial respiration/metabolism</br></br>
</li> PHS gene: Important part of anaerobic bacterial respiration/metabolism</br></br>

Revision as of 02:02, 28 October 2010

iGEM Yale

experimental methods

Our plasmid is composed of three parts: a promoter and a terminator Biobrick as well as a novel addition to the biobrick library, the phsABC gene that is known to encode Thiosulfate Reductase.

(1) phsABC gene and vector
phsABC in pSB74

This central component encodes Thiosulfate Reductase. The gene phsABC was obtained from Dr. Jay Keasling laboratory at University of California, Berkeley. According to their results, Thiosulfate Reductase encoded in the plasmid pSB74 showed the highest activity, so we obtained phsABC from the plasmid pSB74. E. coli DH5α strain were used for plasmid manipulation.

Table from Keasling’s research: comparison of Thiosulfate reductase activity
Figure from Keasling’s research: Sulfide production by phsABC in various plasmids. pSB74 (orange) showed the highest reactivity.
(2) Biobrick Promoter

Promoter used was designed by Caitlin Conboy and was found within the parts registry. This promoter is a Quad Part Inverter: “that is, a PoPS-based inverter composed of four sub-parts: a ribosome binding site, a coding region for a repressor protein (e.g., lambda cI), a terminator, and the promoter (e.g., pLambda) regulated by the encoded repressor protein.” Research into promoter activity by previous groups has suggested that this promoter has a strong on state with a noticeable background in the off state .

(3) Promoter B0034

Biobrick Part:BBa_Q04121.
Length 1370 bp
IPTG-induced (regulatory)

(4) Terminator

Restriction Enzyme Sites:
The sites are shown in New England BioLabs Inc.
Plasmid Construction

The digested sticky ends of the enzymes Xba I and Spe I are complimentary. Once two ends from different combine, neither Xba I nor Spe I can recognize its restriction site in the gene.

By using this ligation method, we added the promoter to phsABC:

Read more about the background of the PHS gene