Cell-free gene expression (CFE) involves activating transcription and translation using crude cellular extracts outside of intact living cells. CFE offers unprecedented control over gene expression and the ability to prototype genetic parts, circuits, pathways, and protein complexes without the constraints of cellular systems. CFE reactions are composed of a cell extract, reaction mixture, and DNA/inducers. Recent advances have overcome challenges like low yields and duration to enable applications like rapid prototyping, biomanufacturing, and development of cell-free biosensors. CFE systems show promise for advancing synthetic biology and building minimal synthetic cells.

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

26. 26