Team:Dundee/Project/ProductionExport

From 2013.igem.org

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    <th>Reaction name</th>
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    <th>Constant</th>
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    <th>Value</th> 
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    <td>Transcription</td>
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    <td>K<sub>Tc</sub></td>
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    <td> 0.03833 nM.s<sup>-1</sup></td>
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    <td>mRNA degradation</td>
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    <td>K<sub>mdeg</sub></td>
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    <td>0.0077 s<sup>-1</sup></td>
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    <td>Translation</td>
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    <td>K<sub>Tl<sub></td>
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    <td>0.25 s-<sup>1</sup></td>
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    <td>PP1 degradation</td>
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    <td>K<sub>pdeg</sub></td>
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    <td>0.00592 s<sup>-1</sup></td>
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Revision as of 16:43, 4 September 2013

iGEM Dundee 2013 · ToxiMop

Dundee 2013

Production & Export

Introduction

The ToxiMop is an engineered E. coli bacteria 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 is because microcystin binds to PP1 in the periplasm.

We explored both the Twin Arginine Translocase (Tat) pathway and 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 transportation, 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 under transportation has a signal sequence attached 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 into the periplasm. The signal peptide is cleaved off and this frees up the TatB-C complex and TatA proteins for further 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.

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

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