Team:EPFL/Description/Lysates
Lysates
Cell-free systems
Synthetic biology has the main goal of creating artificial biological systems that might eventually be used for application in various domains such as medicine or research. Modifying cells to develop new systems is very labour, time intensive and difficult. Synthetic biology approaches problems engineering-driven with design-test cycles, which can still take up considerable amounts of time. Furthermore, the exact behaviour of cells and the influence of interactions between newly engineered and native parts cannot be predicted.
Cell-free systems use all of the inner workings of a cell without having the constricting boundary of the cell wall and thus the precondition of keeping cells alive. This allows speeding the design-test cycles up. When preparing the cell-free systems, all genomic DNA and membranes are eliminated, resulting in a solution containing all of the cells proteins without the limiting factors of a living cell. This system can be used to express proteins for example. Furthermore, having eliminated the membrane of cells also allows bacteria to detect and sense proteins, RNAs and DNAs that would normally not be found in the cytoplasm.
One need of the project was to produce the enzyme β-galactosidase as signaling molecule. Working in lysates was one way to do it. Another possibility was to work in PURExpress, but it is substantially more expensive, thus it was used only with 'delicate' experiments.
According to a paper from Sun et al1, home-made lysate costs approximatively only 0.03$ per μL (depending on the method of cell lysis) instead of 0.79$ for PURExpress. Moreover, they estimated that the production of protein in PURExpress lies at 0.2 mg/ml versu s0.75 mg/ml for home-made lysate, which clearly favours lysate. However, the protein production efficiency can vary between lysate batches, bacterial strains or which protein is produced. PURExpress does have this considerable advantage: The whole environment is a lot more controlled and thus there is less variability with results.
Hence, the first step of the project was to produce functional lysates from different strains of bacteria.
Lysate preparation
Lysates preparation is quite long but the big advantage is that it gives a protein expression system that is cheap and can be lyophilized for better storage. The first step of production is to make a preculture of cells in growth medium (LB or YPTG) incubating overnight. Then, a sample of the preculture must be added again to growth medium in order to have enough bacteria and must be incubated for 4 hours. The next day, the cells are pelleted by centrifugation, washed and sonicated (method based on ultrason in order to break the membrane). Afterwards the solution are again pelleted in order to separate the supernatant - the lysate - from debris. Finally, lysate is extracted from the debris, aliquoted and stored at -80°C. For one good bacterial culture of 200ml, there is usually between 0.5 and 0.7 ml of lysates obtained (gives between 200 and 280 reactions).
Lysate reaction
The next step after lysate production was to make an functional energy solution in order to be able to test the lysates. As lysates is based on using all the working material of cells without keeping them alive, some components that are consumable need to be added. Hence, an energy solution 2 containing amino acids, the four NTPs, tRNA, CoA, NAD, cAMP, salts (magnesium for example) and other things necessary to the cell’s machinery to work is added to the lysate reaction. For an optimized lysate reaction, some buffer is added too.
Finally, DNA, coding for a protein, can be added to the lysate reaction (lysate, energy solution and buffer A) in order to produce protein.
In the project, there is two main expressed proteins: enhanced green fluorescent protein (EGFP) and β-galactosidase. For EGFP, the lysate reaction contains DNA coding for EGFP, lysate, energy solution, buffer A and water for correct final reaction volume. EGFP was used for quantitive reactions in order to determine from which bacteria strains the lysates were the most efficient. For β-galactosidase, the reaction is the same except that the DNA is lacZ (codes for β-galactosidase) and that 5-Bromo-4-Chloro-3-Indolyl β-D-Galactopyranoside (chlorophenol red) must be added. Chlorophenol red is a polysaccharide, which gives a yellow color to the substrate and change to purple when cutted by β-galactosidase. The production of this enzyme was mainly used for qualitative experiment because only few enzymes must be produced in order to have a signal.
Another way to have more sensitive signal was studied: we showed the α-complementation. B-galactosidase is a tetramer of two different subunits: α and Ω parts respectively coded by lacZα and lacZΩ genes. These subunits are non-functional alone, but can be assembled in order to produce a functional β-galactosidase.3 Hence, we decided to produce lysates from bacterial strains that can’t produce the α subunit because of a mutation in lacZα gene and to produce this part afterwards in a lysate reaction. We discovered that these two α and Ω parts can be assembled afterwards in order to produce a functional enzyme. As, the entire β-galactosidase, is a quite long protein to express, the alternative of producing only the α subunit gave us a quicker and more sensitive signal.
We had some problems with the efficiency of protein production. Hence, as lysate contains exonuclease that degrades DNA, we must find out a solution in order to avoid this DNA degradation.
Thus, the next step of the project were to find a solution for this in order to produce more proteins and thus have more sensibility of our diagnostic tool: it was done by adding GamS in the reaction or by using plasmid DNA that cannot be degraded by exonuclease.
GamS
Linear DNA template that is added to a cell-free expression system is at high risk of being cut by an exonuclease still present in the lysate 4. This has as a result that less protein is being produced. When adding the DNA in plasmid form this issue can be prevented.
The GamS protein of phage lambda is an inhibitor of the exonuclease. When added purified to a cell-free system containing linear DNA template, protein expression increases substantially. As purified GamS is quite expensive, another way was explored : this effect could also be achieved when the production of the GamS protein was induced in cells before the subsequent lysis of the cell culture. We showed this by mixing two lysates: M15-T7, which is optimised for protein production in cell-free, and Top10-GamS, which contains GamS protein. Bacterial cells used for Top10-GamS lysate production was done by transformation of Top10 cells with a plasmid coding for the gene GamS.
Lyophilisation
Once cells are lysed and their inner workings extracted, storing the cell-free extract and the energy solution becomes a delicate affair as it has to be stored at -80°C. The necessary infrastructures for this specific storage are easily accessible for scientific laboratories and big enterprises, but can quickly become a problem for countries in development. Cell-free expression systems are a valuable diagnostic tool for viral infections and many more pathogens, so bringing this technique to low-resource regions can be essential. Lyophilisation of the cell-free extracts allows storage at room temperature and only requires a source of nuclease-free water for rehydration.
We have freeze dried our lysates in different combinations and tested their efficiencies upon rehydration.
We tried to optimise the lyophilisation by growing the cells used for lysate production in YTPG medium instead of LB. This YTPG medium permits to make the cells grow using glucose for source of energy and, thus, to use a way simpler and cheaper energy solution. Furthermore, it was demonstrated that YTPG medium permits to have an increased protein production after rehydration of lyophilised lysate reaction.
Lysates
| Lysate | Organism | Strain | RNA polymerase | Antibiotic resistance | Induction | Alpha complementation |
|---|---|---|---|---|---|---|
| BL21(DE3) | E.coli | BL21(DE3) |
|
- | IPTG | No |
| M15 | E.coli | E.coli M15 DZ291 |
|
|
No | Yes |
| M15-T7 | E.coli | E.coli M15 DZ291 |
|
|
IPTG | Yes |
| DH5α | E.coli | DH5α™ T1R |
|
- | No | Yes |
| Top10 | E.coli | Top10 |
|
- | No | Yes |
| Top10-GamS | E.coli | Top10 |
|
|
|
Yes |
The α complementation, as explained before, is the assembly of the two subunits of the β-galactosidase to produce a functional enzyme. Thus, when a bacteria strain is mutated in the gene that code for the α part, this bacteria will not produce the functional β-galactosidase. Hence, this lysate's strain can be used for colorimetric detection as the chlorophenol red is progressively cut by β-galactosidase produced from the DNA added to the lysate. Bacterial strains that contain not the mutation in α part can not be used to study the expression of β-galactosidase by colorimetry as the enzyme is already present in the lysate and will cut immediately all the cholophenol red.
The IPTG induction is for T7 RNA polymerase production during the growth of cells used for lysate production. T7 RNA polymerase is a polymerase that is very specific to his promoter, make less mistakes when synthesis of RNA and produce more RNA. These three advantages motived us to use the T7 promoter for optimised protein expression in lysates.
Hence, we made M15-T7 lysate by transforming M15 cells with plasmid containing the gene for T7 expression for three reasons: first, M15 cells are optimised for protein production in cell free, these cells are mutated in the lacZα part so colorimetric assays were possible and finally the T7 promoter, as explained before is better than the natural present polymerase from e.coli cells.
In the following chapters, we will sometimes use the abbreviation BL21 instead of BL21(DE3) for simplicity.
References
1. Sun, Zachary Z., et al. "Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology." Journal of visualized experiments: JoVE 79 (2013).
2. Kwon, Yong-Chan, and Michael C. Jewett. "High-throughput preparation methods of crude extract for robust cell-free protein synthesis." Scientific reports 5 (2015).
3. Broome, Ann-Marie, et al. "Expanding the utility of β-galactosidase complementation: piece by piece." Molecular pharmaceutics 7.1 (2010): 60
4. Murphy, Kenan C. "The λ Gam protein inhibits RecBCD binding to dsDNA ends." Journal of molecular biology 371.1 (2007): 19-24.
