Team:ZJU-China/Project/GhostSensor/Design

From 2013.igem.org

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.

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