Development of a clostridia-

based cell-free system for

prototyping genetic parts and
metabolic pathways
Group 5
 Background of the Study

Microbes are extremely small living things

that are found all around us and are invisible
to the naked eye. The bioengineering of these
microbes are possible and has great potential
in agriculture, medicine, bioremediation, and
industry.
Background of the Study

This biomanufacturing capability of

bioproducts promises to help address
rapid population growth, an increase in
energy demand, and waste generation.

These conflicts have slowed the

commercialization of innovative
bioproduct manufacturing processes.
 Background of the Study

Clostridia are a group of organisms which

are industrially proven and possess
exceptional substrate, metabolite diversity,
as well as tolerance to metabolic end-
products and contaminants.
Importance of the study
 Methods
Specifically, the goal was to enable cell-free protein
synthesis yields of more than
100 mg/ml by systematically optimizing process
parameters. To achieve this goal,
first, the researchers
streamlined and optimized the extract preparation and
processing procedure. Second, they carried
out a systematic optimization of CFE reaction conditions to
tune the physicochemical environment for stimulating highly
active combined transcription and translation from linear DNA
templates.
 Methods
1. Strains and plasmid constructs.
2. Cell culture and harvest.
3. Extract preparation
4. CFE reaction
5. Quantification of active luciferase
6. Semi-continuous CFE reaction
7. Gas chromatography-mass spectrometry (GS-
MS)
8. Autoradiography
Results
Results
Results
Results
Results
Possible application of the result

Synthetic biology — metabolic

engineering
Biotechnology and
biomanufacturing industries
Platform for non-model
industrially relevant microbes
 Conclusion
The final system was able to produce more than 230 µg/mL of
luciferase within a 3-hour batch reaction, making it one of the more
productive cell-free systems developed to date. C. autoethanogenum
CFE requires unusually high magnesium concentrations. It was
observed that the CFE was compatible with PCR amplicon as
expression templates with minimal purification required. the CFE
system is active enough to produce proteins for building biosynthetic
pathways and assessing their performance in the context of functional
native pathways of cellular metabolism.
Thank You
See You Next Time