Team:ZJU-China/Project/GhostSensor/Background

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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

Ghost Sensor: Background

Interest in developing portable detection devices for on-site analysis has risen sharply in recent years. Many areas, including medical diagnostics, environmental toxicology and bioterrorism controls, would beneﬁt from devices that can perform a rapid ﬁrst-level detection without the need for equipped laboratories or skilled personnel. ZJU-China iGEM team this year successfully built a universal protein detection platform based on the idea of aptamer-induced inner-membrane scaffold dimerization.

Aptamers

Aptamers are functional oligonucleotides that could specifically bind to target molecules and thus are termed chemical antibodies. After nearly 20 years’ endeavor, DNA and RNA aptamers have been identified as binding tightly to a wide range of targets including proteins, peptides, amino acids, drugs, metal ions and even whole cells.

Aptamer-based sensors possess unprecedented advantages compared to biosensors based on natural receptors or antibodies for the following reasons:

1) They can in principle be selected for any given target ranging from small molecules to the whole cells.
2) They can be synthesized with high reproducibility and purity from commercial sources at a low cost which also make them the ideal molecule for gene engineering.
3) DNA aptamers are usually more chemically stable than antibodies or enzymes.

By far, there have been numerous aptamer-based biosensors based on a variety of mechanisms. However, most of them are confined to detect small molecules and strongly rely on the conformational change of aptamers upon binding to targets. ZJU-China this year want to find a quick and robust way to detect large molecules independent of aptamer conformational status.

The idea is simply two things: membrane scaffolds and dimerization. Two aptamers recognizing two different sites of the same target molecule can pull membrane scaffolds together and lead to dimerization. A split GFP or enzyme fused to membrane scaffolds will then come to a whole again and give signal output. However, when it comes to practical design, we have to tackle a series of problems.

E-lysis and bacterial ghost

Because we choose E. coli as our chassis, we don’t want to express our membrane scaffolds in the outer-membrane due to its rigidity and scarcity of proteins. As a result, we express our protein scaffolds in the inner-membrane and make tunnels through inner- and outer-membrane to let target protein freely diffuse into the cell. By this way, we have to make sure that certain level of integrity must be retained or elsewise the tunneled-cells will breakdown into pieces. So we choose to use a simple method for opening cells by PhiX174 protein E-mediated lysis (E-lysis). This lysis procedure is very gentle, avoiding all undesireable side effects of mechanical or chemical procedures to destroy the cell envelope. E-lysis introduces a transmembrane tunnel structure with a diameter of 40–200 nm in the envelope complex of the cells. The hole has fused inner and outer membranes at its orifice, and the driving force for E-lysis is the osmotic pressure difference between the cytoplasm and the medium.

There are two great things about E-lysis. First, it keeps the periplasmic space intact which permits further use of this microenvironment. Second, we can adjust the diameter of the holes simply by including different concentrations of MgSO₄ into the solution, which allow different size of targets ranging from proteins to virus to come in.

The resultant cell envelope is called bacterial ghost. The bacterial ghost platform technology has been used as an innovative system for vaccine, drug or active substance delivery. Using bacterial ghosts as delivery vehicles for subunit or DNA-vaccines the particle structure and surface properties of bacterial ghosts are targeting the carrier itself to primary antigen-presenting cells. Here we render bacterial ghosts a new role as an ideal candidate for membrane scaffold-based biosensor.

Electronic microscopy of bacterial ghost. (A. Witte, et al., Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli, Arch Microbiol (1992) 157:381 388)

Split TEM-1 β-lactamase

Protein fragment complementation assay is a widely used method for protein interaction detection. The two fragments of the protein is rational designed and based on protein interaction assisted folding. TEM-1 β-lactamase is relatively small and monomeric, is well characterized structurally and functionally, can be easily expressed, and is not toxic to prokaryotic cells. Furthermore, no orthologs of β-lactamase exist in many prokaryotes, and thus a protein fragment complementation assay based on β-lactamase could be used universally in prokaryotes without any intrinsic background activity. We split TEM-1 β-lactamase according to [4] into N-terminal consisting of a.a. 26-196 and C-terminal consisting of a.a. 198-290.

Diagram illustrating the split of TEM-1 β-lactamase. (André Galarneau et al., Nature Biotechnology, 2002, 20, 619-22)

The activity of TEM-1 β-lactamase can be detected with a wide range of methods, such as simple colorimetric in vitro assays using the cephalosporin nitrocefin and assays in intact cells using the fluorescent substrate. There are also many commercial immune colloidal gold strips in China to detect β-lactamase in food and beverage. These strips are cheap, sensitive and robust, making them the ideal candidate for large scale applications.

Biotin-avidin interaction

Biotin-avidin interaction has a dissociation constant Kd on the order of 10−15 M, which is one of the strongest known protein-ligand interactions. Biotin is a small chemical which can be modified to DNA or RNA. By biotinylation of our aptamers, we can link aptamers (which is oligonucleotides) to proteins via biotin-avidin interaction. In order to achieve this goal, we fused streptavidin, which is a commonly used tetramer avidin, to the cytoplasmic terminal of our inner-membrane scaffold. So here our aptamer acts like a bridge with its one end attached to target molecules and the other end firmly hold to membrane scaffolds.

Schem illustrating the avidin-biotin interaction. Avidin, streptavidin or NeutrAvidin Protein can bind up to four biotin molecules, which are normally conjugated to an enzyme, antibody or target protein to form an avidin-biotin complex. (Thermo Scientific)

References

[1] Juewen Liu, Zehui Cao, and Yi Lu, Functional Nucleic Acid Sensors, Chem. Rev. 2009, 109, 1948–1998

[2] S. Resch et al., Aqueous release and purification of poly(b-hydroxybutyrate) from Escherichia coli, Journal of Biotechnology 65 (1998) 173–182

[3] Hijiri Hasegawa et al., Improvement of Aptamer Affinity by Dimerization, Sensors 2008, 8, 1090-1098

[4] André Galarneau et al., β-Lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein–protein interactions, nature biotechnology 2002, 20, 619-22

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Next: Ghost Sensor: Design

@@ Line 18: / Line 18: @@
 Aptamers are functional oligonucleotides that could specifically bind to target molecules and thus are termed chemical antibodies. After nearly 20 years’ endeavor, DNA and RNA aptamers have been identified as binding tightly to a wide range of targets including proteins, peptides, amino acids, drugs, metal ions and even whole cells.
-Aptamer-based sensors possess unprecedented advantages compared to biosensors based on natural receptors or antibodies for the following reasons. 1) They can in principle be selected for any given target ranging from small molecules to the whole cells. 2) They can be synthesized with high reproducibility and purity from commercial sources at a low cost which also make them the ideal molecule for gene engineering. 3) DNA aptamers are usually more chemically stable than antibodies or enzymes.
+Aptamer-based sensors possess unprecedented advantages compared to biosensors based on natural receptors or antibodies for the following reasons:
+# 1) They can in principle be selected for any given target ranging from small molecules to the whole cells.
+# 2) They can be synthesized with high reproducibility and purity from commercial sources at a low cost which also make them the ideal molecule for gene engineering.
+# 3) DNA aptamers are usually more chemically stable than antibodies or enzymes.
 By far, there have been numerous aptamer-based biosensors based on a variety of mechanisms. However, most of them are confined to detect small molecules and strongly rely on the conformational change of aptamers upon binding to targets. ZJU-China this year want to find a quick and robust way to detect large molecules independent of aptamer conformational status.
 The idea is simply two things: membrane scaffolds and dimerization. Two aptamers recognizing two different sites of the same target molecule can pull membrane scaffolds together and lead to dimerization. A split GFP or enzyme fused to membrane scaffolds will then come to a whole again and give signal output. However, when it comes to practical design, we have to tackle a series of problems.
 === E-lysis and bacterial ghost ===