User:Cheuk Ka Tong/Sandbox3

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Contents

Cell-Free Systems

Introduction

An important aspect of our project is to investigate the use of cell-free systems (CFS) to realise new potentials for simple constructs. To date, work on synthetic biology has been done using the chassis of bacterial cells. However the use of living, replicating engineered bacteria poses a huge limitation for applications in the food and medicine industries for reasons of public safety. In line with the specifications of our two genetically engeineered machines - Cell by Date and Infector Detector, our team decided to introduce cell-free expression systems as a new chassis to the field of synthetic biology.

In-Vitro vs. In-Vivo Expression Systems

In-Vitro Expression Systems
In-Vivo Expression Systems
Lower pathogenicity because of non-proliferative nature Higher pathogenicity because of proliferative nature
Process is quick and simple requiring only preparation of cell extract and feeding solution and subsequent addition of DNA template Process is laborious involving DNA cloning and transformation and protein expression
Control can be achieved easily using modified reaction conditions such as addition of accessory elements or inhibitory factors Less controllability because of the presence of endogenous substances and because cells do not survive extreme conditions
Both plasmid and linear DNAs and can be used as templates for expression Linear DNAs are easily degraded by endogenous nucleases
Protein degradation is minimized by adding protease inhibitors Synthesized proteins may be degraded by endogenous proteases
Synthesis of toxic proteins is not a problem Synthesis of toxic proteins may kill the cells
Production of proteins containing unnatural amino acids can be achieved Difficult to produce proteins containing unnatural amino acids
Shorter lifespan since system cannot replicate Longer lifespan since system can replicate
More expensive because of the constant need for nutrient and energy supply Less expensive because of the ability of the system to generate energy from relatively cheap nutrient source
Less characterized, less experience of use in the laboratories Better characterized, more experience of use in the laboratories

Types of Cell Extracts

In-vitro synthesis of proteins using cell-free extracts consists of two main processes - transcription of DNA into messenger RNA (mRNA) and translation of mRNA into polypeptides. Coupled transcription-translation systems usually combine a bacteriophage RNA polymerase and promoter (T7, T3, or SP6) with eukaryotic or prokaryotic extracts. In addition, the PURE system is a reconstituted CFS for synthesizing proteins using recombinant elements.

Comparison between different types of cell extracts

Properties
Rabbit Reticulocyte Lysate
Wheat Germ Extract
E. coli Extract
Reconstituted Extract
Types
Eukryotic
Eukryotic
Prokaryotic
Artificial
Uses
Widely used for in-vitro translation
Mostly used for in-vitro translation
Mostly used for coupled transcription-translation
Used for in-vitro translation
Templates
  • mRNAs from viruses or eukryotes
  • Capped or un-capped mRNAs
  • mRNAs from viruses or eukryotes
  • Capped mRNAs
  • Viral mRNAs with stable secondary structures
  • Un-capped mRNAs
  • Plasmid or linear DNAs
  • Capped or uncapped mRNAs
Post-translational modifications
Yes
Yes
No
Yes

Types of Compartmentalization

Previous research has been done to optimize cell extracts for in vitro protein synthesis. Their endogenous genetic content is removed so that exogenous DNAs or mRNAs can be expressed. Nuclease activity has been reduced and degradation of certain amino acids has been identified. ATP regenerating systems have also been added to improve the energy supply. Different strategies of compartmentalization have been explored to prolong the lifespan of CFS.

  1. Batch-mode CFS
    • Transcription-translation reaction is carried out in bulk solution. Expression is usually limited by nutrient (nucleotides and amino acids) and energy supplies.
  2. Continuous-exchange CFS
    • Transcription-translation reaction is separated from feeding solution by a dialysis membrane. Expression is sustained by diffusion of nutrients from the feeding soltuion to the reaction. Wastes generated by the reaction is diluted in the feeding solution.
  3. Liposome-encapsulated CFS
    • The reaction is separated from feeding solution by a phospholipid bilayer. Expression is maintained for a longer time period than batch-mode CFS because of exchange of materials between the reaction and the feeding solution across the membrane. More reliable exchange of materials is established by inserting a non-specific pore protein with a suitable channel size into the phospholipid bilayer.

Achievements

To get around the problem of having bacteria in contact with food or medical devices in the applications of Cell by Date and Infector Detector respectively, we aimed to express reporter DNA constructs within a CFS consisting of cell extract encapsulated in selectively permeable vesicles. Our plan was to first prepare the cell extract and perform in-vitro expression in bulk solution. In parallel, we would investigate and optimize conditions for phospholipid vesicle formation. The final stage of our project would involve combining the two, so that protein expression is achieved inside the vesicles.

In-Vitro Expression

  • Attempted to make S30 E. coli cell extract and feeding solution
  • Successfully used commercial S30 E. coli cell extract and feeding solution from Promega for expression of GFP
  • Successfully used homemade S30 E. coli cell extract with commercial feeding solution for expression of GFP
  • Attempting to characterize the temperature range and timespan of expression of our reporter DNA constructs using commercial S30 E. coli cell extract and feeding solution

Vesicle Formation

  • Successfully formed empty vesicles, as well as vesicles encapsulating GFP, in Tris-Cl buffer
  • Successfully formed vesicles encapsulating GFP in homemade S30 E. coli cell extract
  • Attempting to enclose cell extract in vesicles to attain expression of reporter DNA constructs
  • Attempting to find suitable pore proteins to prolong vesicle and expression lifespan
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