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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 </html>
-== Ghost Sensor: Design ==
+== '''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 place.
+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.
 <center>
-[[File:ZJU-GhostSensor-1.png|600px]]
+[[File:Bacterialghost ppt.jpg|600px]]
-(Timo Langemann et al., The bacterial ghost platform system Production and applications, Bioengineered Bugs, 2010). The two domains of protein E help to fuse inner- and outer-membrane, keeping the periplasmic space intact.
+<small>
+(Protein E is a '''dual-domain''' protein cloned from bacteriophage &psi;X-174. The two domains of protein E help to fuse inner- and outer-membrane, keeping the periplasmic space intact.)
+</small>
 </center>
-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.
-We altogether used two sets of inner-membrane scaffolds, one was based on split EGFP 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.
+[[File:15mer29merthrombinaptamer.jpg|400px|center]]
+<html><div class="clean"></div></html>
-<center>
-[[File:ZJU-GhostSensor-2.png|600px]]
-</center>
-For the first set of membrane scaffolds, two residuals of EGFP (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 pulled together and the two residuals of EGFP will meet each other again 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.
+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-4.png|600px]]
+[[File:ZJU-GhostSensor-2.png|600px]]
-[[File:ZJU-GhostSensor-3.jpg|600px]]
 </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.
+<html>
+<div style="float:left;">Previous:
+  <a href="./Background">Bacterial Ghost: Background</a>
+</div>
+<div style="float:right;">Next:
+  <a href="./Results">Bacterial Ghost: Results</a>
+</div>
-<center>
-[[File:ZJU-GhostSensor-5.png|600px]]
-</center>
-This activity can be detected by commercial β-lactamase testing strips, which are widely used strips in China 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 and robustness.
-<html>
 </div>
    </div>