Investigating a multi-host BioBrick vector and secretion of cellular - PowerPoint PPT Presentation

Investigating a multi-host BioBrick vector and secretion of cellular products

The Team

Team Roster • High School Outreach: 5 High School Students – 2 different local high schools – 1 high school foreign exchange student • 3 Graduate Advisors • 5 Undergraduate Students

Our Home - Utah, USA

Logan, UT, USA

Challenges in Biomanufacturing • Secretion – - Allows cheaper product recovery - Provides basis for continous production • Broad-Host-Range Vectors – - Opens BioBrick library for use in microorganisms other than E. coli Photobioreactor for Biomanufacturing

Broad-Host- Secretion Range Vector

Investigating Secretion: Bioplastics • Polyhydroxyalkanoates (PHAs) – Class of biodegradable plastics – Produced by microorganisms – Useful material properties • Issue: Cost – 50% of cost due to product recovery TEM
image
of
PHB
in
 Ralstonia
 eutropha
 taken
at
Utah
State.



Secretion Based Recovery • Signal Peptides – Target proteins for secretion • Phasin (PhaP) – Proteins associated with PHA granules • Silver fusion

Secretion Objectives: • Create library of BioBricks for secretion – Signal Peptides – Phasin – Green fluorescent protein • Demonstrate functionality of parts

Approach - Parts Needed

Approach - Signal Peptides • Five
signal
pep;des
synthesized
 – Silver
fusion
compa;ble
 • Represent
most
common
 secre;on
pathways
 • Constructed
and
sequenced


Approach – Proteins • Phasin: – Genomic DNA of R. eutropha – Mutation of PstI site • GFP: – New Silver fusion compatible part – Larger Stokes shift (Ex. 395 nm, Em. 509 nm) – Easy to monitor – Similar in size to phasin

Testing GFP • Testing – Fluorescence Emission – SDS-PAGE (27 kDa band) GFP

PHB Production • Plasmid:
 phbCAB 
genes

 • Confirmed
using
 1 H
NMR
 1 H
NMR
of
PHB
in
 E .
 coli. 


Phasin Secretion • GeneIII/phasin
composite
 
 • Analyzed
supernatant

 • Phasin
size:
about
22
kDa
 • Indicates
secre;on


Secretion Broad-Host- Range Vector

Breaking Host Borders Eliminate
constraint
of
BioBrick
library
to
 E.
coli 
 (Photosynthetic Microbe)

pCPP33 • Stable
in
 P.
pu7da
 • Resistance
to
 tetracycline
 • Characterized
 mul;ple
cloning
site


BioBrick Compatibility

Summary of Project Results • Constructed and sequenced: 49 BioBricks – 8 with demonstrated functionality – Some of our favorites: • Secretion of phasin • pCPP33 broad-host chassis converted to BioBrick format – Transferred successfully into R. sphaeroides , P. putida , and Synechocystis PCC6803

Conclusions BioBrick
Borders
were
broken
by
 1. Designing
secre;on
systems
 2. Conver;ng

a
broad‐host‐range
vector
to
BioBrick
format

 Significance
in
biomanufacturing


We would like to thank our Sponsors QUESTIONS?

Genetic Composition of BHR-V • Classed
as
mobilizable
plasmids
 • Contain
some
 tra
 genes
(mob)
 • OriT
(RP4
used
in
this
study)
 • Unstable
without
RP4
helper
plasmid