Team:ULB-Brussels/Project

From 2014.igem.org

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<h1>2A Peptides</h1>
<h1>2A Peptides</h1>
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<p><i>2A sequences</i> are short peptides (corresponding to 18 amino-acids) encoded in some viral genomes. It allows the production of two different proteins from a single open reading frame : one upstream and one downstream of 2A peptide. This cleavage is done by a ribosome skipping between a glycyl residue of the 2A peptide and the next prolyl of the peptidic sequence. The ribosome can then continue to translate the downstream sequence [<b>Fig 4a</b>] into a second, separated protein.  
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<p><i>2A sequences</i> are short peptides (corresponding to 18 amino-acids) encoded in some viral genomes. It allows the production of two different proteins from a single open reading frame : one upstream and one downstream of 2A peptide. This cleavage is done by a ribosome skipping between a glycyl residue of the 2A peptide and the next prolyl of the peptidic sequence. The ribosome can then continue to translate the downstream sequence [<b>Fig 4a</b>] into a second, separated protein. The C-terminal extremity of the upstream protein is fused with the 2A while the N-terminal extremity of the downstream protein is fused with a proline.</p>
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The mechanism of this skipping is not yet understood, but it seems that the nascent peptidic chain binds the ribosome [<b>Fig 4b</b>] and inhbits its peptidyl transferase activity during the incorporation of the glycyl residue [<b>6</b>]. </p>
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<p>The mechanism of this skipping is not yet understood, but it seems that the nascent peptidic chain binds the ribosome [<b>Fig 4b</b>] and inhbits its peptidyl transferase activity during the incorporation of the glycyl residue [<b>6</b>]. </p>
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<br/><font size="1"><b>Figure 4</b>: Cleavage of the 2A peptide. a) Peptidic sequence of a 2A peptide. The skipping occurs between the penultimate (G) and the last (P) residue of the 2A, resulting in two peptidic chains. b) Proposed mechanism for 2A ribosome skipping. The nascent chain incorporates a glycyl residue and binds the ribosome. This interaction results in an inhibition of ribosomal peptidyl transferase activity and an interruption of the peptidic chain [<b>6</b>].</font>
<br/><font size="1"><b>Figure 4</b>: Cleavage of the 2A peptide. a) Peptidic sequence of a 2A peptide. The skipping occurs between the penultimate (G) and the last (P) residue of the 2A, resulting in two peptidic chains. b) Proposed mechanism for 2A ribosome skipping. The nascent chain incorporates a glycyl residue and binds the ribosome. This interaction results in an inhibition of ribosomal peptidyl transferase activity and an interruption of the peptidic chain [<b>6</b>].</font>
</section>
</section>
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<p>2A sequences have been well characterized in eukaryotes but not in bacteria (except for the f2A peptde) [<b>7</b>]. Since these two domains of life have different ribosomal structures, 2A functionnality is still to be analysed in bacteria.</p>
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<p>2A have an advantage over other methods to pair two proteins : it allows a powerful quality control. Indeed, the second peptide cannot be produced if the first one has been subjected to a frameshifting mutation.</p>
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<p>Since the downstream protein does not necessarily begins by a prolin, we added the DNA sequence for the prolin at the end of the gene of the 2A peptide. This ensures that p2A will always be followed by a proline undependantely of the nature of the downstream protein. It is thus this construction of 2A peptide + proline that is referred to as "2A peptide" in the other sections of our project. Hence, the C-terminal extremity of the upstream protein is fused with  p2A, and N-terminal extremity of the downstream protein is fused with the proline we added to the 2A peptide. <p>
<p>Since the downstream protein does not necessarily begins by a prolin, we added the DNA sequence for the prolin at the end of the gene of the 2A peptide. This ensures that p2A will always be followed by a proline undependantely of the nature of the downstream protein. It is thus this construction of 2A peptide + proline that is referred to as "2A peptide" in the other sections of our project. Hence, the C-terminal extremity of the upstream protein is fused with  p2A, and N-terminal extremity of the downstream protein is fused with the proline we added to the 2A peptide. <p>
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A great advantage of the use of p2A over other methods is that it allows a powerful quality control: the antitoxin will be produced only and only if the upstream protein is correctly translated (or punctually muted, which is very rare). Any premature stop codon or “frame-shift” will be detected.</p>
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<!-- This paragraph was yet written (with other sentences, same idea) in one precedent TA paragraph ! -->
There are several 2A-like sequences, but a 2A peptide working well within E.coli could not be found in the literature, exceptely some studies about the 2A peptide <i>F2A</i> [<b>7</b>]. However, the 2A peptide <i>P2A</i> was found to works properly in S.cerevisiae. We thus decided to separate our <i>wetlab in two separate projects</i>: on the first hand, we would try to find a 2A peptide that works in procaryotes (<i>Escherichia coli</i>); on the other hand, we will build the Mighty Coli system in eukaryotes (<i>Saccharomyces cerevisiae</i>) using the P2A. We will thus use two different TA systems: ccdB-ccdA for E.coli, and Kid-Kis for S.cerevisiae (respectively Toxin-Antitoxin).</section>
There are several 2A-like sequences, but a 2A peptide working well within E.coli could not be found in the literature, exceptely some studies about the 2A peptide <i>F2A</i> [<b>7</b>]. However, the 2A peptide <i>P2A</i> was found to works properly in S.cerevisiae. We thus decided to separate our <i>wetlab in two separate projects</i>: on the first hand, we would try to find a 2A peptide that works in procaryotes (<i>Escherichia coli</i>); on the other hand, we will build the Mighty Coli system in eukaryotes (<i>Saccharomyces cerevisiae</i>) using the P2A. We will thus use two different TA systems: ccdB-ccdA for E.coli, and Kid-Kis for S.cerevisiae (respectively Toxin-Antitoxin).</section>

Revision as of 09:58, 12 October 2014

$~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ \newcommand{\MyColi}{{\small Mighty\hspace{0.12cm}Coli}} \newcommand{\Stabi}{\small Stabi}$ $\newcommand{\EColi}{\small E.coli} \newcommand{\SCere}{\small S.cerevisae}\\[0cm] ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ \newcommand{\PI}{\small PI}$ $\newcommand{\Igo}{\Large\mathcal{I}} \newcommand{\Tgo}{\Large\mathcal{T}} \newcommand{\Ogo}{\Large\mathcal{O}} ~$ Example of a hierarchical menu in CSS

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- Université Libre de Bruxelles -


$Introduction$


Retrieved from "http://2014.igem.org/Team:ULB-Brussels/Project"

Purpose and definitions

Production of recombinant proteins by microorganisms such as bacteria (Escherichia coli) or yeasts (Saccharomyces cerevisiae, Pichia pastoris) is a key process in pharmacy (vaccines, insulin) and biotechnology (enzymes, antibodies). The tank in which the protein-producing microorganisms are grown is called a bioreactor. One can define it as a controlled environment where a chemical reaction (in our case, protein synthesis) is catalyzed by micro-organisms.

Heterogeneity in bioreactors

Micro-organisms used to produce recombinant proteins in bioreactors are often seen as a homogeneous population. However, stressed subpopulations may appear, resulting in reduced quantity and quality of the production. Indeed, stressed cells consume nutrients and space but have a reduced productivity [1]. Several factors may induce heterogeneity in a population : desynchronisation in cell cycle phases, emergence of mutants or local variations in physico-chemical conditions within the reactor [2]. Getting rid of these stressed subpopulations might be an effective way to increase both quality and quantity of production in bioreactors.

The Mighty Coli insight

We thought that the underlying problem of population heterogeneity is that micro-organisms do not have any advantage to produce a protein of interest (PI). Such production would be a unnecessary constraint for the cell,

We thus decided to give microorganisms some incentive to overproduce the PI. That would be done by killing the non-productive bacteria, or that would enter a stressed physiological state.

We used two genetic compounds to apply this principle and design Migthy Coli : the toxin-antitoxin systems (TA) and the 2A peptides.

Toxin-Antitoxin Systems

TA systems are operons made of two linked genes encoding respectively for a stable toxic protein and an unstable inhibitor of this toxin : an antitoxin. TA systems naturally occur in mobile genetic elements such as plasmids and are used to maintain these in a microbial population. When a bacterium possessing such a plasmid divides, the generated daughter cells might not inherit a copy of the plasmid due to a stochastic partition. In this case, the antitoxin, unstable, is quickly degraded, allowing the toxin to perform its function and kill the daugther cell in a process known as post-segregational killing (PSK) [Fig. 1]. This system allows a plasmid to be selected and maintained in a bacterial population even if it does not confer any advantage for the host. Therefore, TA systems can be seen as selfish entities, virtually making bacteria addicted to them [3].



Figure 1 : PSK and plasmid addiction. Cells that inherit a plasmid encoding a TA system can grow normally. As there is no system to part plasmids equivalently in daughter cells, some cells might not receive a plasmid during division. Such cells would still have toxins and antitoxins in their cytosol but these will not be renewed due to the loss of TA genes. Antitoxins are often unstable and quickly degraded by a protease under these conditions, leaving the toxins free and able to kill the cell, effectively eliminating cells losing plasmids that encode TA systems.

TA systems can be divided into three groups, depending on the nature and mode of action of its components [3]. We will mainly use type II TAs in which both components are proteins and the antitoxin binds to the toxin, preventing it from performing its function [Fig. 2]. Toxin functions and structures in type II TA systems are diversified, allowing us to chose how cells will die when stressed. Two systems will be used to illustrate our project : CcdBA and Kid/Kis.



Figure 2 : Type II TA systems. The toxin is a protein that inhibits a vital function of the cell (translation, replication, peptidoglycan synthesis, etc.) and the antitoxin is another protein that binds to this toxin, preventing it from causing harm to the cell. This antitoxin is often unstable and subject to a quick degradation. Should the transcription of the TA operon stops, the antitoxin would swiftly be degraded, allowing the toxin to kill or damage the cell. Type II TA operons are negatively regulated at the transcriptionnal level by their respective TA complexes.

CcdBA and the DNA gyrase

CcdBA is the most studied type II TA system, an operon coming from the E. coli fertility factor (F plasmid). CcdB is the toxin and CcdA the antitoxin. CcdB is an inhibitor of the DNA gyrase, an enzyme that negatively supercoils circular DNA, such as bacterial chromosomes and plasmids. In the majority of the organism, the DNA is negatively supercoiled, but during transcription or replication, DNA gets positively supercoiled due to ADN/ARN polymerase activity. The role of gyrase is to reduce the linking number of the DNA by relaxing positive supercoiled DNA. CcdB associates with the DNA gyrase subunit that binds to DNA and inhibits its activity when it is bound to DNA, resulting in DNA double strand breaks due to the progession of polymerases during replication or transcription [Fig. 3]. It causes the activation of emergency signals and possibly death of the cell [4].



Figure 3 : DNA gyrase mechanism & CcdB poisoning. a : The DNA gyrase is a tetramer made of two GyrA and two GyrB subunits. b : An undefined DNAsegment (G-segment) can bind between two GyrB subunits. c : Another segment (T-segment) enters the GyrB clamps. d : Upon ATP biding, a conformationnal change occurs. It induces a double strand break in the G-segment and closes the GyrB clamps e : CcdB binds to GyrA subunits. h : CcdB prevents the gyrase from joining G-segment ends, resulting in DNA double strand breaks. f : The T-segment is released on the other side of the G-segment by an transitionnal state of GyrA subunits. g : ATP is hydrolysed to ligate the G-segment and to return the complex to its original conformation. The complex can start a new catalytic cycle of supercoiling.

The Kid/Kis system

The Kid/Kis system (also known as PemK/PemI) is a TA system that stabilizes the R100 plasmid, a plasmid that confers resistance to various compounds such as mercury or antibiotics. Kid is a toxin with endoribonuclease activity [5]. When the Kis antitoxin is not expressed, the Kid toxin inhibits cell growth and metabolism by cleaving mRNAs. Unlike CcdBA, the Kid/Kis system is functional in eukaryotes, making it suitable for developping MightyColi in S. cerevisiae.

Why TA systems ?

We want to build a biological system selecting only highly productive cells. By coupling the production of the PI with an antitoxin in a strain expressing the corresponding toxin, we hope that stressed cells experiencing a drop in PI productivity will be killed by the ensuing drop in antitoxin production. There are many ways to couple the production of two proteins, f.e. polycistronic mRNAs in procaryotes or internal ribosome entry sites (IRES). We chosed to use a 2A peptide to pair the PI and the antitoxin.


2A Peptides

2A sequences are short peptides (corresponding to 18 amino-acids) encoded in some viral genomes. It allows the production of two different proteins from a single open reading frame : one upstream and one downstream of 2A peptide. This cleavage is done by a ribosome skipping between a glycyl residue of the 2A peptide and the next prolyl of the peptidic sequence. The ribosome can then continue to translate the downstream sequence [Fig 4a] into a second, separated protein. The C-terminal extremity of the upstream protein is fused with the 2A while the N-terminal extremity of the downstream protein is fused with a proline.

The mechanism of this skipping is not yet understood, but it seems that the nascent peptidic chain binds the ribosome [Fig 4b] and inhbits its peptidyl transferase activity during the incorporation of the glycyl residue [6].



Figure 4: Cleavage of the 2A peptide. a) Peptidic sequence of a 2A peptide. The skipping occurs between the penultimate (G) and the last (P) residue of the 2A, resulting in two peptidic chains. b) Proposed mechanism for 2A ribosome skipping. The nascent chain incorporates a glycyl residue and binds the ribosome. This interaction results in an inhibition of ribosomal peptidyl transferase activity and an interruption of the peptidic chain [6].

2A sequences have been well characterized in eukaryotes but not in bacteria (except for the f2A peptde) [7]. Since these two domains of life have different ribosomal structures, 2A functionnality is still to be analysed in bacteria.

2A have an advantage over other methods to pair two proteins : it allows a powerful quality control. Indeed, the second peptide cannot be produced if the first one has been subjected to a frameshifting mutation.

Since the downstream protein does not necessarily begins by a prolin, we added the DNA sequence for the prolin at the end of the gene of the 2A peptide. This ensures that p2A will always be followed by a proline undependantely of the nature of the downstream protein. It is thus this construction of 2A peptide + proline that is referred to as "2A peptide" in the other sections of our project. Hence, the C-terminal extremity of the upstream protein is fused with p2A, and N-terminal extremity of the downstream protein is fused with the proline we added to the 2A peptide.

There are several 2A-like sequences, but a 2A peptide working well within E.coli could not be found in the literature, exceptely some studies about the 2A peptide F2A [7]. However, the 2A peptide P2A was found to works properly in S.cerevisiae. We thus decided to separate our wetlab in two separate projects: on the first hand, we would try to find a 2A peptide that works in procaryotes (Escherichia coli); on the other hand, we will build the Mighty Coli system in eukaryotes (Saccharomyces cerevisiae) using the P2A. We will thus use two different TA systems: ccdB-ccdA for E.coli, and Kid-Kis for S.cerevisiae (respectively Toxin-Antitoxin).

Stability of Mighty Coli

The regulation of natural TA system can be complex, and is not always well understood. Since we want our system to be as simple, as safe and as productive as possible, we decided to use well characterized promoters for our system. We thus decided to synthetize first the toxin and the antitoxin from different plasmids [Fig 5], each under the control of a different promoter and each bearing a different antibiotic resistance gene. The TA system is thus used to boost protein production and the plasmid stability is assured by antibiotic resistance.


Figure 5 : Distinction between Toxin and Antitoxin Vectors: the plasmid on the left containing the toxin gene (when not added in the bacterial chromosome) and the second plasmid (regulated by the T7 constitutive promoter) containing the protein of interest (RFP/GFP marker) and the antitoxin gene.

Aim

The aim of our project is thus double. On the one hand, we would try to build Mighty Coli into two different chassis (E.coli and S.cerevisiae), and characterize the quantity and quality of their protein production. On the other hand, we will test several 2A peptide sequences to find one that would work efficiently in E.coli.

Bibliography

  • [1] H.M. Davey & D.B. Kell, (1996). Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses, Microbiological reviews, 60(4), 641-696.
  • [2] S. Müller, H. Harms & T. Bley, (2010). Origin and analysis of microbial population heterogeneity in bioprocesses, Current opinion in biotechnology, 21(1), 100-113.
  • [3] F. Hayes & L. Van Melderen, (2011). Toxins-antitoxins: diversity, evolution and function, Critical Reviews in Biochemistry and Molecular Biology, 46(5), 386-408.
  • [4] M.H. Dao-Thi, L. Van Melderen, E. De Genst, H. Afif, L. Buts, L. Wyns & R. Loris, (2005). Molecular basis of gyrase poisoning by the addiction toxin CcdB, Journal of molecular biology, 348(5), 1091-1102.
  • [5] J. Zhang, Y. Zhang, L. Zhu, M. Suzuki, & M. Inouye (2004). Interference of mRNA function by sequence-specific endoribonuclease PemK, J. Biol. Chem., 279(20), 20678-20684.
  • [6] V.A. Doronina, P. de Felipe, C. Wu, P. Sharma, M.S. Sachs, M.D. Ryan & J.D. Brown, (2008). Dissection of a co-translational nascent chain separation event. Biochemical Society Transactions, 36(4), 712-716.
  • [7] 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.
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