Difference between revisions of "Team:Calgary/Secretion"

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<p>We ultimately chose a secretion based system, due to the impact of its advantages in space; a system that requires no chemical input, no replacement of bacteria, and provides continuous production saves both time and room aboard the spacecraft, both of which are precious commodities during space travel.</p>
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<p>We decided to implement a PHB-secretion system that takes advantage of the naturally-occurring Type I Hemolysin system found in <i>E. coli</i> Intracellular PHB granules are tagged with the hemolysin secretion motif, then the granule is secreted from the cell via hemolysin membrane transport proteins. This system is advantageous for our space application because it reduces extra materials/power needs, allows cells to remain intact for easier PHB separation from the liquid media, and only minimal bacteria replacement would be required. The hemolysin secretion mechanism has been used previously by the <a href="https://2016.igem.org/Team:SDU-Denmark/Experiments#secretion">SDU-Denmark 2016</a> and <a href="https://2009.igem.org/Team:Utah_State/Secretion">Utah 2009</a> iGEM teams for PHB secretion. We have used <a href="http://parts.igem.org/Part:BBa_K2018024">Part: BBa_K2018024</a> from the SDU-Denmark 2016 as the basis for the design of our secretion complex.</p>
  
 
<h2>Type 1 Secretion</h2>
 
<h2>Type 1 Secretion</h2>
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<p>PHB is secreted through the hemolysin pathway, a type 1 secretion system, that is endogenous to <i>E. coli</i>. Naturally, hemolysin toxin (HlyA) is secreted from the <i>E. coli</i> as a defense mechanism. This is a single step process involving three main proteins: HlyB, HlyD and TolC. HlyB is an active transport protein that uses ATP in the cytoplasmic membrane, HlyD is a membrane fusion protein that spans the periplasm and connects the inner and outer membrane, and TolC rests in the outer membrane. When HlyB recognizes the C-terminus of HlyA, it stimulates formation of the secretion channel and the toxin is secreted (Thomas et al., 2014).</p>
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<h2>Our Secretion System</h2>
 
<h2>Our Secretion System</h2>
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<p>To use this system in our biologically engineered cells, we added the C-terminus of HlyA to the end of a phasin molecule. Phasin, coded for by PhaP, is a small structural protein found in bacteria that naturally produce PHB. It binds to intracellular PHB, and therefore the PHB granules with bound phasin-HlyA tag are secreted as one (Rahman et al., 2013).</p>
  
 
<p><center><img src="https://static.igem.org/mediawiki/2017/0/08/Calgary2017_SecretionGraphic1.png" alt="Secretion Pathway" style="width:100%"></center></p>
 
<p><center><img src="https://static.igem.org/mediawiki/2017/0/08/Calgary2017_SecretionGraphic1.png" alt="Secretion Pathway" style="width:100%"></center></p>

Revision as of 04:48, 26 October 2017

Header

Secretion

Overview

After the E. coli BL21 (DE3) engineered by the Synthesis subgroup has produced polyhydroxybutyrate (PHB), it is stored as intracellular granules that range in size from 60-80nm. This creates a challenging problem: efficient extraction of the desired PHB from inside the cell. During the early stages of our project, we explored many possible PHB extraction methods. Before deciding on using a PHB-secretion system, we evaluated the advantages and disadvantages of each system in terms of our space application.

Method:

Secretion

Chemical Extraction

Heat-Induced Lysis

Description:

Secretion of PHB via the Type I Hemolysin secretion pathway, which is endogenous to E.coli (Rahman et al., 2013).

Traditionally used solvent extraction, using chemicals such as chloroform and sodium hypochlorite (Hahn et al., 1994).

T4 lysis genes (holins and endolysins) under the control of Lambda promoter, pR with thermosensitive repressor proteins, cI587. When heated, lysis genes become activated.(George et al., 1987).

Advantages:

  • No chemicals are required
  • PHB can be removed from media without killing cells
  • Secretion is automatic
  • Easier separation of PHB from media because there is minimal amounts of cellular debris
  • Continuous production process

  • Efficient yields
  • Simple

  • No chemicals required
  • Lysis is automatic

Disadvantages:

  • Lower recorded yield than other methods
  • Less characterized in literature than lysis mechanisms

  • Transport of chemicals to and from Mars is high cost
  • Requires manual input of chemicals
  • Lysis kills cells, (would need to ship stocks of our engineered E. coli to Mars)
  • Cellular debris from lysis makes separation of PHB more difficult

  • Heating our fermenters on Mars would use huge amounts of power
  • Lysis kills cells, (would need to ship stocks of our engineered E. coli to Mars
  • Cellular debris from lysis makes separation of PHB more difficult

We ultimately chose a secretion based system, due to the impact of its advantages in space; a system that requires no chemical input, no replacement of bacteria, and provides continuous production saves both time and room aboard the spacecraft, both of which are precious commodities during space travel.

We decided to implement a PHB-secretion system that takes advantage of the naturally-occurring Type I Hemolysin system found in E. coli Intracellular PHB granules are tagged with the hemolysin secretion motif, then the granule is secreted from the cell via hemolysin membrane transport proteins. This system is advantageous for our space application because it reduces extra materials/power needs, allows cells to remain intact for easier PHB separation from the liquid media, and only minimal bacteria replacement would be required. The hemolysin secretion mechanism has been used previously by the SDU-Denmark 2016 and Utah 2009 iGEM teams for PHB secretion. We have used Part: BBa_K2018024 from the SDU-Denmark 2016 as the basis for the design of our secretion complex.

Type 1 Secretion

PHB is secreted through the hemolysin pathway, a type 1 secretion system, that is endogenous to E. coli. Naturally, hemolysin toxin (HlyA) is secreted from the E. coli as a defense mechanism. This is a single step process involving three main proteins: HlyB, HlyD and TolC. HlyB is an active transport protein that uses ATP in the cytoplasmic membrane, HlyD is a membrane fusion protein that spans the periplasm and connects the inner and outer membrane, and TolC rests in the outer membrane. When HlyB recognizes the C-terminus of HlyA, it stimulates formation of the secretion channel and the toxin is secreted (Thomas et al., 2014).

Our Secretion System

To use this system in our biologically engineered cells, we added the C-terminus of HlyA to the end of a phasin molecule. Phasin, coded for by PhaP, is a small structural protein found in bacteria that naturally produce PHB. It binds to intracellular PHB, and therefore the PHB granules with bound phasin-HlyA tag are secreted as one (Rahman et al., 2013).

Secretion Pathway

Our Construct

Secretion Construct

Experiments

Future Directions

Works Cited