Team:Imperial College London/Modules/Fast Response

The third module of our bacterial detector consists of a fast response reporter module that upon an input signal arrival gives out a yellow signal output visible by the naked eye. Traditional reporters in biochemical research usually rely on transcription-translation of a specific reporter molecule (such as Green Fluorescent protein) which is time consuming and/or might involve sophisticated equipment for its detection. In this module we introduce a novel reporter system where expression of a low molecular weight protease allows this protease to act on an existing pool of substrate. Due to this double amplification step, the output signal should be several orders of magnitude faster than traditional reporters.

Below is a quick overview of our system, followed by a more in-depth analysis of the system.

Catechol (2,3) dioxygenase (C23O) is the protein product of the XylE gene. It originates from Pseudomonas putida and the active protein is made up of a homotetramer of C2,3O 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.

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 recognised and cleaved by TEV protease. This amino acid 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 no longer sterically 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.

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 has a relatively low molecular weight (242 aa) compared to other protease candidates available. This means that the synthesis of the protease will be relatively quick. For more information about TEV, please see the PDB file with the 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?

• Due to the small size of the TEV gene, it is likely that the time taken for TEV transcription and translation will be very short and so 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-C2,3O fusion protein) is an inactive form of the chosen output enzyme Catechol 2,3 dioxygenase. Since TEV protease is a relatively fast enzyme, it has a high turnover rate of substrate molecules per unit of time. This is AMPLIFICATION STEP #1.

• The now activated C2,3O is an enzyme itself of a huge turnover rate. As a consequence, a vast number of catechol molecules would be converted to yellow colored product per unit of time. This is AMPLIFICATION STEP #2.

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.

[1] An archetypical extradiol-cleaving catecholic dioxygenase: the crystal structure of catechol 2,3-dioxygenase (metapyrocatechase) from Pseudomonas putida mt-2 Akiko Kita1, Shin-ichi Kita2, Ikuhide Fujisawa1, Koji Inaka3, Tetsuo Ishida4, Kihachiro Horiike4, Mitsuhiro Nozaki4 and Kunio Miki5,