SCIENTIFIC PRESENTATION ON BIOTECHNOLOGY, GENETIC EXPRESSION

1. The extended world of
CELL-FREE GENE EXPRESSION
PRESENTED BY:
BAISHALI TAMULI
Integrated MSc 6th semester
BBI17008

2. INDEX
1.Introduction
2. Pros of CFE
3. Overcoming challenges of CFE
4. An overview of CFE reactions
5. Cell-free probing of cellular functions
• Prototyping genetic parts
• Prototyping genetic circuits
• Cell-free versus cellular prototyping
• Prototyping enzymatic pathways
• Building cells
6. Applications of cell free systems
7. Conclusion 2

3. What is cell free biology?
• Cell free biology : activation of biological processes without the use of
intact living cells
• Recent technical renaissance has brought a breakthrough evolution in
various arenas.
Massive opportunities to
• Transform synthetic biology
• new approaches to design of synthetic gene networks
• On demand Biomanufacturing
• building cells from bottom up etc
Introduction
3

4. INTRODUCTION…
What is Cell Free gene Expression (CFE)?
• Activation of transcription and translation using crude cellular extracts
• In vitro protein synthesis using cell extracts and exogenous resources
• Huge implication of cell biology
4

5. INDEX
1.Introduction
2.Pros of CFE
3.Overcoming challenges of CFE
4.An overview of CFE reactions
5.Cell-free probing of cellular functions
• Prototyping genetic parts
• Prototyping genetic circuits
• Cell-free versus cellular prototyping
• Prototyping enzymatic pathways
• Building cells
6.Applications of cell free systems
7.Conclusion 5

6. Pros of CFE
• Eliminate the constraint of sustaining life
• Unprecedented control over gene expression
• Direct manipulation of the experimental system
• Bypass limitations on molecular transport across cell wall
• Focus resource utilization on distinct networks
• Biosynthesis of a single product
6

7. INDEX
1.Introduction
2.Pros of CFE
3.Overcoming challenges of CFE
4.An overview of CFE reactions
5.Cell-free probing of cellular functions
• Prototyping genetic parts
• Prototyping genetic circuits
• Cell-free versus cellular prototyping
• Prototyping enzymatic pathways
• Building cells
6.Applications of cell free systems
7.Conclusion 7

8. Overcoming challenges of CFE
Challenges:
• Low and variable protein synthesis yields
• Short reaction duration
• Reagent cost
• Small reaction scale
• Inability to correctly fold complex protein and protein assemblies
• Inability to control reaction environment
Technical renaissance made possible:
• Protein yields exceeding grams of protein produced per litre of reaction volume
• Batch reactions last for more than 10 hours
• Reaction scale reached 100-litre milestone
• Less expensive and more accessible
• Protocols simplified
8

9. INDEX
1.Introduction
2.Pros of CFE
3.Overcoming challenges of CFE
4.An overview of CFE reactions
5.Cell-free probing of cellular functions
• Prototyping genetic parts
• Prototyping genetic circuits
• Cell-free versus cellular prototyping
• Prototyping enzymatic pathways
• Building cells
6.Applications of cell free systems
7.Conclusion 9

10. An Overview of CFE reactions
Prepared from three components:
The cell extract
+
A reaction mixture
+
A mixture of DNA and inducers
Source: Silverman et.al, nature reviews genetics,2019
10

11. INDEX
1.Introduction
2.Pros of CFE
3.Overcoming challenges of CFE
4.An overview of CFE reactions
5.Cell-free probing of cellular functions
• Prototyping genetic parts
• Prototyping genetic circuits
• Cell-free versus cellular prototyping
• Prototyping enzymatic pathways
• Building cells
6.Applications of cell free systems
7.Conclusion 11

12. 12
Cell-free probing of cellular functions
1. Biochemical study of cellular functions
2. Ability to prototype
• Individual genetic parts
• Complex genetic designs
• Biosynthetic pathways
• Protein complexes
3. Designing artificial minimal cells

13. Prototyping Genetic parts
13
Source: Silverman et.al,nature reviews genetics,2019
• Rapid assay of performance of
individual genetic parts
• Designing new genetic parts like
promoters and RBS
• Libraries of genetic parts created
without the need for
• plasmid construction
• transformation
• colony picking

14. Prototyping genetic circuits
• Analyzing how genetic parts
function together in synthetic
genetic control networks
• Contribution of each part can
be easily manipulated
• Optimizations of parts to
predict how a system would
function in cells
14
Source: Silverman et.al, nature reviews genetics,2019

15. Cell –free versus cellular prototyping
• Cell free prototyping faster to
prototype non model and slow
growing organisms.
• Provide improved control over
physiochemical environment.
• The extent to which cell-free
experiments replicate cellular
results is not well defined.
Fig: expression of a template through cellular and cell free experiment
15
Source: Silverman et.al, nature reviews genetics,2019

16. Enzymatic pathway prototyping
• Reconstruction using CFE of individual pathway enzymes
• High degree of flexibility to model the kinetics and stability of individual enzymes
• A great promise for prototyping the synthesis of biofuels, high value commodity chemicals.
Fig: reconstruction of enzymatic pathways
16
Source: Silverman et.al, nature reviews genetics,2019

17. 1. Protein-protein interactions
Screened in high throughput
using combinatorial mixing
Protein complex and assembly prototyping
2. Application in functional genomics
Cellular system level characterization of protein function in vitro
17
Source: Silverman et.al, nature reviews genetics,2019

18. Building cells
• Design and study of synthetic cells integrating multiple genetic and metabolic pathways.
• Recapsulating cellular functions by encapsulating CFE systems and synthetic gene circuits
inside liposomes.
• Mimicking cellular behaviors for energy generation, vesicular communication and
organelle formation.
18
Source: Silverman et.al, nature reviews genetics,2019

19. INDEX
1.Introduction
2.Pros of CFE
3.Overcoming challenges of CFE
4.An overview of CFE reactions
5.Cell-free probing of cellular functions
• Prototyping genetic parts
• Prototyping genetic circuits
• Cell-free versus cellular prototyping
• Prototyping enzymatic pathways
• Building cells
6. Applications of cell free systems
7.Conclusion 19

20. Applications of cell free systems
1.Design of cell free biosensors
• CFE reactions can be lyophilized and shipped
• They can maintain their activity for months.
• The reactions produce a fluorescent or colorimetric output when supplied
with target analyte
20
Source: Silverman et.al, nature reviews genetics,2019
a. Point of use cell free biosensing

21. 21
b. Nucleic acid sensors
Applications of cell free systems…
Design of cell free biosensors…
• Detection of disease causing viruses and bacteria
• CFE reactions produce reporter protein only in presence of target sequence.
Source: Silverman et.al, nature reviews genetics,2019

22. 2.Cell-free Biomanufacturing
22
Applications of cell free systems…
• Using cold chain independent freeze dried lysate
• DNA and CFE reagents transportable
• Upon rehydration and mixing, produce a wide variety of proteins and
chemicals
Source: Silverman et.al, nature reviews genetics,2019

23. INDEX
1.Introduction
2.Pros of CFE
3.Overcoming challenges of CFE
4.An overview of CFE reactions
5.Cell-free probing of cellular functions
• Prototyping genetic parts
• Prototyping genetic circuits
• Cell-free versus cellular prototyping
• Prototyping enzymatic pathways
• Building cells
6.Applications of cell free systems
7.Conclusion 23

24. Conclusions and future directions
24
• CFE has made enormous technical gains over 5-10 years
• An increasing number of non model strains are becoming available
• Challenge of prolonging cell free metabolism needs to be overcome
• Uncovered insights into genetic mysteries
• CFE expanding to new and unexpected application spaces to address
global challenges

25. 25
Questions
and
Discussion

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