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

Photobioreactor for 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

Secretion Broad-Host- 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

GFP

Testing GFP

• Testing

– Fluorescence Emission – SDS-PAGE (27 kDa band)

1H
NMR
of
PHB
in
E.
coli.


PHB Production

• Plasmid:
phbCAB
genes


• Confirmed
using
1H
NMR


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


QUESTIONS?

We would like to thank our Sponsors

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