Team:Yale/Our Project/Methods

From 2010.igem.org

(Difference between revisions)
Line 92: Line 92:
<br />
<br />
-
<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 phsABC gene</a>
<div style="display:none;" id="background">
<div style="display:none;" id="background">
<p>
<p>
-
</li> PHS gene: Important part of anaerobic bacterial respiration/metabolism</br></br>
+
</li> phsABC gene: Important part of anaerobic bacterial respiration/metabolism</br></br>
• Studied since 1970's; several plasmids found (ie: phs pEB40, pSB103, pSB77, pSB107 etc)</br></br>
• Studied since 1970's; several plasmids found (ie: phs pEB40, pSB103, pSB77, pSB107 etc)</br></br>
• Thiosulfate reductase is a transmembrane protein involved in the second step of the Sulfate-Reducing Bacteria pathway of thiosulfate reduction. Thiosulfate reductase catalyzes the dissimilatory reduction of inorganic thiosulfate to hydrogen sulfide and sulfite. </br></br>
• Thiosulfate reductase is a transmembrane protein involved in the second step of the Sulfate-Reducing Bacteria pathway of thiosulfate reduction. Thiosulfate reductase catalyzes the dissimilatory reduction of inorganic thiosulfate to hydrogen sulfide and sulfite. </br></br>

Revision as of 02:23, 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 through Addgene from Dr. Jay Keasling's laboratory at University of California, Berkeley. According to their results, thiosulfate reductase encoded in the plasmid pSB74 showed the highest activity catalytic activity, so we obtained phsABC from the plasmid pSB74.

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 .


Promoter B0034

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


(3) Biobrick Terminator The terminator used was the 129 bp BBa_B0015 designed by Reshma Shetty. It is actually a double terminator composed of BBa_B0010 and BBa_B0012 and the BioBrick assembly scar & was chosen for its reliability and availability.

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, inserted phsABC into B0015:



Read more about the background of the phsABC gene