I. WetLab Structure

The design of the $\MyColi$ system requires several intermediate constructions and experiments which will be explained on this page.

II. E.coli Chassis

Our system is built on the property of post-transcriptional cleavage of the 2A peptide. It allows us to subordinate the production of the protein of interest to the production of the antitoxin. It was reported that peptide sequences from the 2A family have different efficiency in prokaryotic cells than in eukaryotic cells. Hence, we will test the efficiency of several 2A sequences to find the best for our system.

A. Screening of the post transcriptional cleavage activity of different 2A-like sequences in E.coli

In order to make a screening of the post transcriptional cleavage activity of different 2A peptides in E.coli, we designed two strategies:
$\hspace{0.25cm}$ - A positive selection of bacteria where the cleavage occurs by using a RFP::2A::phoA fusion.
$\hspace{0.25cm}$ - A negative selection of bacteria where the cleavage occurs by using a RFP::2A::ccdB fusion.

1. Positive selection using RFP:: 2A::phoA

Alkaline phosphatase (phoA) is a periplasmic enzyme whose activity is easily detectable, even at low level, on chromogenic substrate (XP-medium (5-bromo-4-chloro-3-indolyl phosphate)). It is usually used to study protein secretion, but we will use it as a molecular marker for the activity of the 2A peptide. If phoA is correctly synthetized and exported in the periplasm, the substrate acquires a characteristic blue color. (Hoffman and Wright 1985; van Geest and Lolkema 2000)

In order to assess the efficacity of the 2A peptid, we need to design a plasmid containing 2 molecular markers (the red fluorescent protein (RFP) and phoA separated by a 2A peptide (RFP::2A::phoA). After cloning this plasmid in bacteria lacking the phoA gene in their genome (those bacteria were obtained from E.coli Keio Knockout collection) and after growth on chromogenic and selective XP-medium, we should be able to observe 4 types of results:
$\hspace{0.25cm}$ 1. Colourless colonies and blue medium
$\hspace{0.25cm}$ 2. Colourless colonies and colourless medium
$\hspace{0.25cm}$ 3. Red colonies and blue medium
$\hspace{0.25cm}$ 4. Red colonies and colourless medium.

The only interesting case is the third because the PI is functional and the rapporteur protein is active. However, some checks must be made. In fact, two different control groups will be needed for this experiment.

Indeed, the N-terminal extremity of phoA is a signal sequence that allows it to be translocated into the bacterial periplasm, where it will be folded in its active form. (Hoffman and Wright 1985; van Geest and Lolkema 2000) It means that the addition of the prolin from the 2A peptide to the N-extremity of phoA (Luke, 2012) could possibly disrupt the translocation process. If such a phenomenon should occur, our positive screening would reveal only negative results for phoA, even when the 2A peptide works correctly. We must thus design this control experiment in order to check that the translocation of phoA in the periplasm occurs even if we add a prolin on its N-extremity. The control experiment should produce one of the following results:
$\hspace{0.25cm}$ - Colonies on blue medium
$\hspace{0.25cm}$ - Colonies on colourless medium.

If proline::phoA bacteria colour their medium, it will mean that the alkaline phosphatase can be translocated into the periplasm even if it is fused with an additional proline on its N-extremity. The previous experiment (Screening of different phoA-like sequences) will thus be carried on. If their medium remains colourless, it means that we cannot use phoA to test the activity of the 2A peptides and we will have to re-design an experiment with other molecular markers. The control group of this control experiment will consist of bacteria lacking the phoA gene in which we will have inserted the phoA gene ourselves, and it should produce bacteria that colour their XP-medium.

2. Negative selection using RFP::2A::ccdB

This second screening will serve as an independent test of the first screening. CcdB is a prokaryotic toxin affecting the DNA gyrase, which leads to the death of the cell. (Van Melderen, 2010) After growth on selective medium, the construction RFP::2A::ccdB expressed by our bacteria should lead to 3 kinds of results:
$\hspace{0.25cm}$ 1. Red colonies
$\hspace{0.25cm}$ 2. Colourless colonies
$\hspace{0.25cm}$ 3. No colonies.

In the 1$^{st}$ case, the 2A peptide would not have allowed the translation of the downstream protein (ccdB) but the upstream protein (RFP) would be correctly translated. In 2$^{nd}$ case, the translational cleavage would do not have occured, resulting in the fusion of RFP and ccdB and in their respective inhibition. In the 3$^{rd}$ case, the translational cleavage would have occurred and ccdB would have been correctly translated, resulting in the cell death.

The control group of this experiment is a plate of bacteria possessing a RFP::ccdB recombinant plasmid. It is necessary to verify that the toxin is no functional when the protein is fusionned with RFP. The protein become functional only after the cleavage of the 2A peptide allowing a negative selection.

B. Characterization of the Mighty coli system in E.coli

1. Analysis of the improvement in the quantity of the product

The quantification of yield improvement shall be done by spectrophotometry in collaboration with F. Delvigne from the University of Liège (Ulg), using scale-down reactors to reproduce in the lab the conditions occurring within large bioreactors. (Delvigne et al., 2009) We will compare the GFP production yield of a common E.coli bacterium and the one of an E.coli expressing the Mighty Coli system (one plasmid containing the ccdB gene, and the other containing the construction GFP::2A::ccdA).

2. Analysis of the efficiency to reduce the heterogeneity of population

If we have the time, it will be done with the collaboration F. Delvigne from the University of Liège (Ulg), using scale-down reactors to reproduce in the lab the conditions occurring within large bioreactors. (Delvigne et al., 2009)

III. S.cerevisiae Chassis

A. P2A peptide cleavage rate - Modelling

The modelling team needs to know the cleavage rate of the P2A peptide in order to compute the effectiveness of Mighty Coli. It will also give us quantitative expectation of the empiric measurement, which could lead to interesting axis of research if the measurement is too different from the prediction.

It will be done by the construction of a insert linking the P2A to two molecular markers: GFP and RFP (GFP::P2A::RFP). After ligation, electroporation and growth on selective medium, we will thus be able to measure by spectrophotometry the GFP/RFP ratio as well as the production rate of both proteins, which should be a good indicator of those same data in Mighty Coli.

B. Characterization of the Mighty coli system in S.cerevisiae

1. Analysis of the improvement in the quantity of the product

The quantification of yield improvement shall be done by spectrophotometry in collaboration with F. Delvigne from the University of Liège (Ulg), using scale-down reactors to reproduce in the lab the conditions occurring within large bioreactors. (Delvigne et al., 2009) We will compare the GFP production yield of a common yeast and the one of a yeast expressing the Mighty Coli system (one plasmid containing the Kid gene, and the other containing the construction GFP::P2A::Kis).

2. Analysis of the improvement in the quality of the product

To evaluate the improvement in the quality of the protein production, we will use Apol1 as protein of interest. Indeed, this protein possesses several isoforms, each of them the resulting of a mutation of the original Apol1 gene, and the concentration of each can be easily measured. We will thus compare the relative concentrations of the isoforms of Apol1 produced by a common yeast with those of a yeast expressing the Mighty Coli system (one plasmid containing the Kid gene, and the other containing the construction Apol1::P2A::Kis).

Since all the frameshift mutations affecting the plasmid containing Apol1 will also disrupt the translation of the antitoxin, we expect the mutated forms of Apol1 to be far less produced by the Mighty Coli yeasts.

IV. Constructions and Biobricks Summaries

In order to complete our project, we will need to build 11 recombinant plasmids (6 in $\EColi$, 5 in $\SCere$). Each chassis consists in an independent project, which should enable us to complete at least one of them at the end of the summer.

Figure 1 $:\hspace{0.16cm}$ Constructions summary.

At the end of our project, we should have sent at least 7 biobricks, and maybe more if the screening of the different 2A peptides is positive.

Figure 1 $:\hspace{0.16cm}$ Biobricks summary.

Lab Protocols

Electroporation

Dyalisis (with 0.0250µm filter) for 20 minutes of 12µl of ligation solution and 12µl of digested plasmid.

Place 50µl of electrocompetent bacteria in an cold electroporation cell (don't touch the electrodes). Inject the dyalisis product into the eletrocompetent cell.

Insert the electrocompetent cell into the electroporation machine, and electroporate at 250V. Without spark and if the time constant approximates 4.6, all go well.

PCR Amplification

Miniprep: GenElute™ Plasmid Miniprep Kit

Bacterial cells are harvested via centrifugation, subjected to a modified alkaline-SDS lysis procedure and the DNA adsorbed onto silica in the presence of high salts. Contaminants are then removed by a simple wash step. Bound DNA is eluted in water or Tris-EDTA buffer.

Gel Purification: GenElute™ Gel Extraction Kit

The GenElute Gel Extraction Kit combines silica-binding technology with the convenience of a spin or vacuum column format. DNA fragments of interest are extracted from slices of an agarose gel and are bound to a silica membrane. Contaminants are removed by a simple spin or vacuum wash. The bound DNA is then eluted.

The purified DNA is suitable for a variety of downstream applications, such as automated DNA sequencing, PCR, restriction digestion, cloning, and labeling.

Column Purification: GenElute™ PCR Clean-Up Kit

The GenElute PCR Clean-Up Kit combines the advantages of silica binding with a microspin format. The DNA is bound on a silica membrane within the spin column. The bound DNA is washed and the clean, concentrated DNA is eluted in the buffer of choice. Each column can purify up to 100 μL or 10 μg of PCR amplified DNA and recover up to 95% of PCR products between 100 bp and 10 kb. More than 99% of the primers and most primer-dimers (< 40 bp are removed).

PCR Cloning: Clontech™ In-Fusion HD Cloning Plus

The In-Fusion Enzyme premix fuses PCR-generated sequences and linearized vectors efficiently and precisely, utilizing a 15 bp overlap at their ends. This 15 bp overlap can be engineered by designing custom primers for amplification of the desired sequences. This method can be used to clone single or multiple fragments into a single vector without subcloning.

Bibliography

[5] G.A. Luke, (2012). Translating 2A research into practice, Innovations in Biotechnology, E.C. Agbo ed., ISBN: 978-953-51-0096-6, InTech Croatia, 161-186.

[6] M.D. Ryan, M.L.L. Donnelly, A. Lewis, A.P. Mehrotra, J. Wilkie & D. Gani, (1999). A model for Nonstoechiometric, Co-translational Protein Scission in Eukaryotic Ribosomes. Bioorganic Chemistry, 27, (Feb 1999), pp55-79, ISSN: 0045-2068.

[7] M. van Geest & J.S. Lolkema, (2000). Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals, Microbiol. Mol. Biol. Rev. March 2000 vol. 64 no. 1 13-33.

[8] F. Delvigne, M. Boxus, S. Ingels & P. Thonart, (2009). Bioreactor mixing efficiency modulates the activity of a prpoS::GFP reporter gene in E.coli, Microbial Cell Factories 2009, 8:15.