Team:Dundee/Project/ProductionExport

Introduction

The ToxiMop is an engineered E. coli bacterium that expresses PP1 and can be used as a molecular mop to remove microcystin from contaminated water. Central to successfully engineering this machine was PP1 production and export. This was crucial as the microcystin binding interaction predominantly takes place with PP1 in the periplasm.

We explored both the Twin Arginine Translocase (Tat) and General Secretory (Sec) pathways as potential export routes for PP1. However, initial Western blot results indicated that PP1 was exported into the periplasm much more successfully via the Tat pathway than via Sec. Therefore, production and export based on Tat transport, was selected as a modelling focus to allow us to optimise the construction of our prototype ToxiMop.

Building a Model for Tat Transport

The Tat machinery is a biological pathway that transports folded proteins from the cytoplasm into the periplasm. It consists of three small membrane proteins; TatA, TatB and TatC.

TatB and TatC together form a TatB-C complex. The protein destined for transport has a signal sequence at its N-terminus which is recognised by and binds to the TatB-C complex. This positions the protein ready for export. TatA proteins then polymerise and form a ring structure surrounding the protein allowing it to penetrate the membrane and pass into the periplasm. The signal peptide is cleaved off and this frees up the TatB-C complex and TatA proteins for further transport.

Figure 1: Processes involved in Tat transport.

Tat-dependent Transport of PP1

PP1 has a molecular mass of 37kDa. Assuming that PP1 is spherical, it would require 20 TatA proteins to form a ring large enough to accommodate it and enable it to penetrate the membrane [1]. We define this structure as a TatA assembly.

For transport, PP1 in the cytoplasm (PP1_cyto) binds to TatB-C, forming a PP1 TatB-C complex (PP1B-C). The TatA assembly then surrounds the PP1 TatB-C complex. We define this product as PP1_export. PP1_export is then exported into the periplasm (PP1_peri), releasing the TatA assembly and TatB-C back into the membrane to assist in further rounds of transport.

Making the following assumptions:

• TatA assemblies are pre-formed from TatA proteins
• PP1 exported to the periplasm remains in the periplasm
• all other processes are reversible

Figure 2: Ring structures formed by polymerisation of TatA proteins

We arrive at this framework to describe Tat transport of PP1.

Production

Before we can transport any PP1, we first need to produce the protein. This involved inserting the PP1-encoding gene into a plasmid vector and transforming the plasmid into host cells. These cells then expressed the gene.

We consider the transcription and translation required for this gene expression. This simple production scheme is derived by assuming that both mRNA and protein can degrade. Due to the heterologous nature of PP1, its degradation constant is particular significant.

Using the law of mass action and appropriate rate constants, we create a mathematical system that represents each reaction. These values and equations are shown below:

Reaction name	Constant	Value
Transcription	KTc	0.03833 nM.s-1
mRNA degradation	Kmdeg	0.0077 s-1
Translation	KTl	0.75 s-1
PP1 degradation	Kpdeg	0.0192 s-1

Figure 3: Table 1 - PP1 production rate constants [2].

Our team developed a series of MATLAB programs to solve the models which we considered. The code for these programs along with further analysis is available at this repository [3]. The program v1_odes_solver_PP1Production solves this system numerically.

The production model predicts that we will produce approximately 1200 net PP1 in the cytoplasm of each E. coli cell. This gives us initial indications of the amount of PP1 we can produce per cell and allows us to determine how many cells are required to mop up fixed concentrations of microcystin. We use this information to examine the practicality of the ToxiMop. For example, if we assume all the PP1 is exported to the periplasm and exploit the one-to-one binding of microcystin with PP1, then 0.6g of cells are required to clean up one litre of contaminated water that is classified as unsafe by World Health Organisation (WHO) regulations. The production of this mass of cells is highly achievable for our Wet team to produce.

Figure 4: PP1 production graph - Figure 5: 1173 PP1 are produced per cell division

Production & Export

Combining our separate schemes for protein production (2) and Tat transport (1), we built a model that describes PP1 Production & Export.

Reaction name	Constant	Value
Transcription	KTc	0.03833 nM.s-1
mRNA degradation	Kmdeg	0.0077 s-1
Translation	KTl	0.75 s-1
PP1 degradation	Kpdeg	0.0192 s-1
Recognition binding	K1	8E3 M-1.s--1
Recognition unbinding	K2	8E3 M-1.s--1
Assembly association	K3	200E4 M--1.s--1
Assembly disassociation	K4	0.00167 s-1
Export	K5	10 s-1

Figure 6: Table 1 - PP1 production & export rate constants [2].