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Team:Imperial College/Waste Degradation: SRF
From 2013.igem.org
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<p>Are you planning an iGEM project with E.coli that involves protein secretion? You are at the best place for finding all the information you need for making it happen.</P> | <p>Are you planning an iGEM project with E.coli that involves protein secretion? You are at the best place for finding all the information you need for making it happen.</P> | ||
| - | E.coli is commonly used as a chassis in innovative iGEM project that aim to prove the concept and make the case for a novel function in a biologically engineered machine. In our case, we aim to degrade and synthesise plastic and the degradation part of our system needs to be extacellular. There are not only such biotechnological but also many other reasons for the extracellular targeting of a protein: It can be a product with a medical function, like the [www.2009.igem.org/Team:METU-Gene/EGF_Transportation EGF hormone in a previous iGEM project] or be important in bioremediation such as in [https://2013.igem.org/Team:Dundee/Project/Mop Dundee Team’s toximop] system and much more. There are many strategies for secretion. On the following page, you can read about these and find out why we chose the pelB tag for all our degradation enzymes and how we improved the ESTCS2 esterase by changing the phoA outer-membrane anchoring tag in the original Biobrick to pelB. Our story will help you to choose a secretion strategy best suited to your project and also give you guidance with the technical details of using it. | + | E.coli is commonly used as a chassis in innovative iGEM project that aim to prove the concept and make the case for a novel function in a biologically engineered machine. In our case, we aim to degrade and synthesise plastic and the degradation part of our system needs to be extacellular. There are not only such biotechnological but also many other reasons for the extracellular targeting of a protein: It can be a product with a medical function, like the [www.2009.igem.org/Team:METU-Gene/EGF_Transportation EGF hormone in a previous iGEM project] or be important in bioremediation such as in [https://2013.igem.org/Team:Dundee/Project/Mop Dundee Team’s toximop] system and much more. There are many strategies for secretion. On the following page, you can read about these and find out why we chose the pelB tag for all our degradation enzymes and how we improved the ESTCS2 esterase by changing the phoA outer-membrane anchoring tag in the original Biobrick to pelB. Our story will help you to choose a secretion strategy best suited to your project and also give you guidance with the technical details of using it. </p> |
| - | Alternative strategies for protein secretion in E.coli: | + | <p>Alternative strategies for protein secretion in E.coli:</p> |
N terminal secretion-signal peptides are recognised by the sec pathway (1). The pathway transports to the periplasm and additional mechanism are usually necessary to further export the protein to the extracellular space (2). An approach for using this pathway is the addition of an N terminal signal peptide to the target protein which can obtain as high as 90% efficiency in secretion to the extracellular space (3). Such signals are pelB and phoA tags which are available as biobricks. PelB can get cleaved off by pelB peptidase in the periplasm but phoA will anchor your protein to this localisation. | N terminal secretion-signal peptides are recognised by the sec pathway (1). The pathway transports to the periplasm and additional mechanism are usually necessary to further export the protein to the extracellular space (2). An approach for using this pathway is the addition of an N terminal signal peptide to the target protein which can obtain as high as 90% efficiency in secretion to the extracellular space (3). Such signals are pelB and phoA tags which are available as biobricks. PelB can get cleaved off by pelB peptidase in the periplasm but phoA will anchor your protein to this localisation. | ||
Fusion partners are endogenous proteins that naturally get secreted in E.coli and can be fused to the target protein. Some such proteins were demonstrated to be a powerful carriers of medically relevant human proteins in E.coli (4). The yebF and ompF proteins exit the cell via the sec pathway and use additional mechanism for leaving the periplasm where they interact with outer-membrane porins for extracellular secretion (5). The osmY is available as a Biobrick and we have submitted ompF this year. The porin proteins such as ompF and ompA, can be used as fusion partners too and anchor proteins to the outer surface of E.coli. | Fusion partners are endogenous proteins that naturally get secreted in E.coli and can be fused to the target protein. Some such proteins were demonstrated to be a powerful carriers of medically relevant human proteins in E.coli (4). The yebF and ompF proteins exit the cell via the sec pathway and use additional mechanism for leaving the periplasm where they interact with outer-membrane porins for extracellular secretion (5). The osmY is available as a Biobrick and we have submitted ompF this year. The porin proteins such as ompF and ompA, can be used as fusion partners too and anchor proteins to the outer surface of E.coli. | ||
Revision as of 12:42, 17 September 2013
Making Mixed Waste Useable
Overview
Specification
Modelling
Design
Secretion Pathways in E.Coli
Are you planning an iGEM project with E.coli that involves protein secretion? You are at the best place for finding all the information you need for making it happen.
E.coli is commonly used as a chassis in innovative iGEM project that aim to prove the concept and make the case for a novel function in a biologically engineered machine. In our case, we aim to degrade and synthesise plastic and the degradation part of our system needs to be extacellular. There are not only such biotechnological but also many other reasons for the extracellular targeting of a protein: It can be a product with a medical function, like the [www.2009.igem.org/Team:METU-Gene/EGF_Transportation EGF hormone in a previous iGEM project] or be important in bioremediation such as in [https://2013.igem.org/Team:Dundee/Project/Mop Dundee Team’s toximop] system and much more. There are many strategies for secretion. On the following page, you can read about these and find out why we chose the pelB tag for all our degradation enzymes and how we improved the ESTCS2 esterase by changing the phoA outer-membrane anchoring tag in the original Biobrick to pelB. Our story will help you to choose a secretion strategy best suited to your project and also give you guidance with the technical details of using it.Alternative strategies for protein secretion in E.coli:
N terminal secretion-signal peptides are recognised by the sec pathway (1). The pathway transports to the periplasm and additional mechanism are usually necessary to further export the protein to the extracellular space (2). An approach for using this pathway is the addition of an N terminal signal peptide to the target protein which can obtain as high as 90% efficiency in secretion to the extracellular space (3). Such signals are pelB and phoA tags which are available as biobricks. PelB can get cleaved off by pelB peptidase in the periplasm but phoA will anchor your protein to this localisation. Fusion partners are endogenous proteins that naturally get secreted in E.coli and can be fused to the target protein. Some such proteins were demonstrated to be a powerful carriers of medically relevant human proteins in E.coli (4). The yebF and ompF proteins exit the cell via the sec pathway and use additional mechanism for leaving the periplasm where they interact with outer-membrane porins for extracellular secretion (5). The osmY is available as a Biobrick and we have submitted ompF this year. The porin proteins such as ompF and ompA, can be used as fusion partners too and anchor proteins to the outer surface of E.coli. ABC transporters use ATP for transport of specific proteins across the bacterial membrane (6). The ABC transporter of Erwinia chrysanthemi can be expressed in E.coli and export proteins that contain the LARD1 domain as a C terminal fusion (7). The system is biobricked in two separate plasmids (transporter, LARD1) with different antibiotic resistance and it is important to use both at the same time. Pilli-like mechanism is a very innovative secretion strategy which has not been used in an iGEM project before. ?. Iain, what was that thing we saw at the conference? The [http://parts.igem.org/Protein_domains/Localization page in the registry that lists localisation tags] was incomplete and contains eukaryotic and prokaryotic localisation signals mixed together. We have updated this page and, put together the tables below for you to help choose the secretion strategy that if the best fit for your project.| enzyme | source organism | biobrick | reference |
|---|---|---|---|
| EstCS2 | uncultured unknown bacterium (GU256649.1) | BBa_K1149002 | http://www.microbialcellfactories.com/content/10/1/41 [Kang et.al 2011) |
| pueA | Pseudomonas chlororaphis | [http://parts.igem.org/Part:BBa_K1149003 BBa_K1149003] | [http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2000.tb09056.x/pdf (Stern et al., 2000)] |
| pueB | Pseudomonas chlororaphis | [http://parts.igem.org/Part:BBa_K1149004 BBa_K1149004] | [http://www.sciencedirect.com/science/article/pii/S0964830501000427 (Howard et al., 2001)] |
| pudA | Comamonas acidovorans | [http://parts.igem.org/Part:BBa_K1149005 BBa_K1149005] | [http://www.sciencedirect.com/science/article/pii/S0964830598000663 (Allen et al. 1999)] |
| pulA | Pseudomonas fluorescens | [http://parts.igem.org/Part:BBa_K1149006 BBa_K1149006] | [http://www.sciencedirect.com/science/article/pii/S0964830598000687 (Vega et al., 1999)] |
