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Team:Dundee/Project/ProductionExport
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<h2>Introduction</h2> | <h2>Introduction</h2> | ||
| - | <p>The ToxiMop is an engineered <i>E. coli</i> | + | <p>The ToxiMop is an engineered <i>E. coli</i> bacterium that produces 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 is because we anticipate that microcystin binds to PP1 in the periplasm. <br><br> |
| - | We explored both the Twin Arginine Translocase (Tat) pathway and | + | |
| + | We explored both the Twin Arginine Translocase (Tat) pathway and general secretory (Sec) pathway as potential export mechanisms. However, initial Western blot results indicated that PP1 was exported into the periplasm more successfully via the Tat pathway. 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. | ||
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<h2>Building a Model for Tat Transport</h2> | <h2>Building a Model for Tat Transport</h2> | ||
| - | <p>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. | + | <p>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 export 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 cross into the periplasm. The signal peptide is cleaved off and this frees up the TatB-C complex and TatA proteins for further transport. | |
| - | TatB and TatC together form a TatB-C complex. The protein | + | |
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Revision as of 08:22, 1 October 2013
Production & Export
Introduction
The ToxiMop is an engineered E. coli bacterium that produces 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 is because we anticipate that microcystin binds to PP1 in the periplasm.
We explored both the Twin Arginine Translocase (Tat) pathway and general secretory (Sec) pathway as potential export mechanisms. However, initial Western blot results indicated that PP1 was exported into the periplasm more successfully via the Tat pathway. 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 export 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 cross 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 Transport of PP1
Based on its molecular mass of 37kDa, PP1 requires a structure of 20 TatA proteins to enable it to penetrate the membrane [1]. We define this structure as a TatAConstruct.
For transportation, PP1 in the cytoplasm (PP1cyto) binds to TatB-C, forming a PP1 TatB-C complex (PP1B-C). The TatAConstruct then surrounds the PP1 TatB-C complex. This product is defined as PP1export. PP1export is then exported into the periplasm (PP1peri), releasing the TatAConstruct and TatB-C back into the membrane to assist in further transport.
Making the following assumptions:
- TatAConstructs are pre-formed from TatA proteins
- PP1 exported to the periplasm remains in the periplasm
- All other processes are reversible
Figure 2: Ring structure formed by TatA proteins
We arrive at this framework to describe Tat transport of PP1.
Production
However before we could export any PP1, we first needed to produce it. This involved inserting the PP1 gene into a plasmid vector and transforming it into host cells, which in turn expressed the gene. We consider the transcription and translation required for this gene expression. This simple transcription and translation scheme is derived by assuming that both mRNA and proteins can degrade.
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 | 0.25 s-1 |
| PP1 degradation | Kpdeg | 0.00592 s-1 |
