Team:ZJU-China/Project/GhostSensor/Design

From 2013.igem.org

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Latest revision as of 01:51, 29 October 2013

Overview

Bacterial Ghost

Background Design Results

The Ghost Kit

Ghost Sensor Ghost Sponge Ghost Shell The Ghost Kit

Characterization

Background Results

Safety

Overview Artificial Shell E.coli Suicide Safety Form

Parts

Overview BBa_K1054000 BBa_K1054001 BBa_K1054002 BBa_K1054003 BBa_K1054007 BBa_K1054008 BBa_K1054009 BBa_K1054010 BBa_K1054011 BBa_K1054012 BBa_K1054013 BBa_K1054014 BBa_K1054015 BBa_K1054016 BBa_K1054017 BBa_K1054018 BBa_K1054019 BBa_K1054020 BBa_K1054021 BBa_K1054022 BBa_K1054023

Bacterial Ghost: Design

We put Gram-negative bacteria lysis gene protein E under the control of pBAD promoter because of its short inducing time and L-arabinose concentration dependent trait. By making tunnels through inner and outer membrane of E.coli, we solve the conundrum that large molecules cannot enter the cells. At the same time, the periplasmic space remains intact, renders us the chance to utilize it as reaction site.

(Protein E is a dual-domain protein cloned from bacteriophage ψX-174. The two domains of protein E help to fuse inner- and outer-membrane, keeping the periplasmic space intact.)

We try to detect, purify and store thrombin as our proof-of-concept experiment. There have been two well characterized aptamers, one is 15-mer and the other is 29-mer, all show very high and specific affinity to thrombin. We then connected the two aptamers with different length of poly-T linkers, namely short and long linkers, to their 5’ end. At last, all aptamers were modified with 5’ biotin in order that they can bind to streptavidin.

We then built inner-membrane scaffolds consisted of three fusion parts. The split proteins were linked to the periplasmic ends of scaffolds and streptavidin to the cytoplasmic ends. We chose phosphatidylglycerol:: prolipoprotein diacylglyceryl transferase (Lgt) as our transmembrane protein because its function has been well identified by 2012 SJTU-BioX-Shanghai iGEM team. ssDsbA is the signal recognition particle (SRP)-dependent signaling sequence of DsbA. ssDsbA-tagged proteins are exported to the periplasm through the SRP pathway. With ssDsbA fused to the periplasmic terminal, fusion proteins with Lgt are expected to be anchored onto inner membrane of E.coli.

Previous: Bacterial Ghost: Background

Next: Bacterial Ghost: Results

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-We try to detect '''thrombin''' as our proof-of-concept experiment. There have been two well characterized aptamers, one is '''15-mer''' and the other is '''29-mer''', all show very high and specific affinity to thrombin. We then connected the two aptamers with different length of '''poly-T''' linkers, namely short and long linkers, to their 5’ end. At last, all aptamers were modified with '''5’ biotin''' in order that they can bind to streptavidin.
+We try to detect, purify and store '''thrombin''' as our proof-of-concept experiment. There have been two well characterized aptamers, one is '''15-mer''' and the other is '''29-mer''', all show very high and specific affinity to thrombin. We then connected the two aptamers with different length of '''poly-T''' linkers, namely short and long linkers, to their 5’ end. At last, all aptamers were modified with '''5’ biotin''' in order that they can bind to streptavidin.
 [[File:15mer29merthrombinaptamer.jpg|400px|center]]
@@ Line 30: / Line 30: @@
-We altogether used two sets of inner-membrane scaffolds, one was based on '''split GFP''' and the other '''split β-lactamase'''. Our inner-membrane scaffolds consisted of three fusion parts. The split proteins were linked to the periplasmic ends of scaffolds and streptavidin to the cytoplasmic ends. We chose phosphatidylglycerol:: prolipoprotein diacylglyceryl transferase (Lgt) as our transmembrane protein because its function has been well identified by 2012 SJTU-BioX-Shanghai iGEM team. ssDsbA is the signal recognition particle (SRP)-dependent signaling sequence of DsbA. ssDsbA-tagged proteins are exported to the periplasm through the SRP pathway. With ssDsbA fused to the periplasmic terminal, fusion proteins with Lgt are expected to be anchored onto inner membrane of E.coli.
+We then built inner-membrane scaffolds consisted of three fusion parts. The split proteins were linked to the periplasmic ends of scaffolds and streptavidin to the cytoplasmic ends. We chose phosphatidylglycerol:: prolipoprotein diacylglyceryl transferase (Lgt) as our transmembrane protein because its function has been well identified by 2012 SJTU-BioX-Shanghai iGEM team. ssDsbA is the signal recognition particle (SRP)-dependent signaling sequence of DsbA. ssDsbA-tagged proteins are exported to the periplasm through the SRP pathway. With ssDsbA fused to the periplasmic terminal, fusion proteins with Lgt are expected to be anchored onto inner membrane of E.coli.
 <center>
 [[File:ZJU-GhostSensor-2.png|600px]]
-</center>
-For the '''first set''' of membrane scaffolds, two residuals of GFP (BBa_K738006, BBa_K738007) were fused to the periplasmic ends and streptavidin were fused to the cytoplasmic ends. When 19-mer and 25-mer '''biotin-modified''' aptamers are added to the solution and thrombin is present, aptamers act like a '''bridge''' linking streptavidin-fused membrane scaffolds and thrombin. In this way, two molecules of membrane scaffolds are dragged together and the two residuals of GFP will approach each other in close proximity to become an '''integrity part''' capable of giving green fluorescence. With this design, it is believed that we can detect the existence of any protein through fluorescence.
-<center>
-[[File:Zjuscaffold2.jpg|600px]]
-<small>
-(The upper figure illustrates the construction of the second set of membrane scaffold. We cloned the FA- and FB-scaffold genes into pCDFDuet-1 vector to obtain optimal expression. )
-</small>
-</center>
-For the '''second set''' of membrane scaffolds, we want to see the results with naked eyes instead of fluorescence to expand its application in everyday life. So we fused two residuals of '''TEM-1 β-lactamase''' (BBa_K1054010, BBa_K1054011) to the periplasmic ends. Likewise, when 19-mer and 25-mer biotin-modified aptamers are added to the solution and thrombin is present, aptamers act like a '''bridge''' linking streptavidin-fused membrane scaffolds and thrombin. In this way, two molecules of membrane scaffolds are pulled together and the two residuals of β-lactamase will meet each other again to become an integrity part with enzymatic activity (in this case, ampicillin resistance).
-<center>
-[[File:Zjuscaffold1.jpg|600px]]
-<small>
-(The upper figure illustrates the construction of the second set of membrane scaffold. We cloned the BLF1- and BLF2-scaffold genes into pCDFDuet-1 vector to obtain optimal expression.)
-</small>
-</center>
-<html><div class="clean"></div></html>
-This activity can be detected by commercial β-lactamase testing strips, which are widely used strips in China initially designed to detect the residence of β-lactamase in drinking milk. One mechanism of these strips are based on immune colloidal gold and we chose it because of its high sensitivity, robustness and relatively low cost.
-<center>
-[[File:ZJU-GhostSensor-6.JPG|600px]]
-(The upper figure shows
 </center>