Team:Imperial College London/Modules/Fast Response

The third module of our bacterial detector consists of an fast response reporter module that upon input signal arrival it gives out a yellow optical signal output observable by naked eye. Traditional reporters in biochemical research usually rely on transcription-translation of a specific reporter molecule (ex. Green Fluorescence protein) which is time consuming and/or might involve sophisticate equipment for its detection. In this module we introduce a novel reporter system where expression of a small peptidase that acts on a pre-existing pool of substrate as well as a double amplification step should result in an output signal several orders of magnitude faster than traditional reporters. Below an quick overview of our system can be found, followed by a more in depth analysis of the system.

This figure illustrates the point that Catechol-2,3-dioxygenase enzyme is active only when it tetramerises

Catechol-2,3-dioxygenase (also known as C23O) is the protein product of XylE gene. It originates from Pseudomonas putida bacterium and the active protein is made of a homotetramer of monomers. The enzyme catalyses the conversion of a colourless substrate (catechol or substituted catechols) into a bright yellow product (2-hydroxymuconic semialdehyde) within seconds of substrate addition.

GFP is N-terminally added to Catechol-2,3-dioxygenase enzyme so that it does not allows formation of the tetramer by steric hindrance effect. This means that the enzyme is inactive in its monomer form

In our project, we take advantage of the fact that Catechol-2,3-dioxygenase is only active as a tetramer as the active site forms as a result of intersubunit and interface interactions after oligomerization. We have constructed a gene product that encodes a fussion protein. This fusion protein is made of GFP fused to the N-terminal of Catechol-2,3-dioxygenase. The GFP does not allow Catechol-2,3-dioxygenase monomers to associate by steric hindrance, so that the active tetramer is not formed. Instead the fusion protein remain as monomers in the cytosol of the cell.

Detection of the parasite would result in an input signal in the form of the phosphorylated transcription factor ComE. This induces expression of TEV protease, which cleaves fusion protein to activate the system

A very important feature of the fusion protein is its potential for induction of the system. A closer look at the fusion gene construct reveals the secret of the inducibility of our system. The GFP gene is fused to the XylE gene through a protein sequence that is susceptible to TEV protease. This protein sequence is recognized by the active site of the TEV protease so that when TEV is present in the cell, GFP is cleaved off Catechol-2,3-dioxygenase monomers. The free monomers are not longer stericly hindered by the GFP and are now able to associate with each other. Tetramerization of the monomers produces the active Catechol-2,3-dioxygenase enzyme, which in the presence of catechol substrate produces a yellow color.

The GFP-XylE gene construct

Our GFP-XylE gene construct has also some other addition features, which make it easier to work with. To start with it contains an N-terminal His tag which would assist in purification and for in vitro characterization. The same applies to the flag tag sequence which can be recognized by monoclonal antibodies for easier detection and purification. The linker sequence is there to ensure that TEV recognition sequence is readily accessible to the protease for cleavage.

TEV protease is a natural polyprotein cutting viral protease. In our system ComE transcription factors bind to their native ComCDE promoter which was engineered as the promoter for TEV protease. This means that TEV protease is only transcribed and translated upon detection of the Schistosoma parasite.

Why TEV protease? This enzyme has been used extensively in protein engineering as it has a number of key qualities including a high specificity (ENYLFQG), and importantly for our system it is relatively tiny (242 aa) compared to other protease candidates available. Below is can be visualized as it has been deposited in the PDB with accession number 1Q31; TEV protease expression in E.Coli (and consequently B .subtilis) is made efficient by codon optimisation and inducing expression of additional tRNAs.

So what are the key features that make our system a novel example of a fast response mechanism?

• TEV gene due to its small size, is likely that the time that takes for its transcription and translation will be very short will not introduce limiting kinetic factors within our fast response system.
• TEV protease would then act upon a pre-existing pool of inactive substrate already present in the cell. This substrate (GFP-C(2,3)O fusion protein) is an inactive form of the chosen output enzyme Catechol 2,3 dioxygenase. Since TEV protease is an enzyme, it has a big turnover rate of substrate molecules per unit of time. This is AMPLIFICATION STEP #1.
• The now activated C(2,3)O is an enzyme itself of a huge turnover rate. That means that a vast number of catechol molecules would be converted to yellow colored product per unit of time. This is AMPLIFICATION STEP #2.

For our fast response module we have taken advantage of the tetramerization feature which produces the functioning C230 enzyme; the active site forms as a result of intersubunit and interface interactions after oligomerization;

After observing C23O in Pymol, and in addition information obtained from Kita et al which shows that the projecting loop is needed for dimerization and that this is very near to the N-terminus, we chose to engineer (by straightforward DNA synthesis) GFP plus linker (GGGSGGGS ENYLFQG) onto the N-termini of our C230 monomers.

Below is the reaction performed by our systems enzyme:

Image taken from ftp://ftp.genome.jp/pub/kegg ; Where catechol is the colourless substrate converted by ring cleavage into 2 hydroxymuconate semialdehyde.