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Team:INSA Toulouse/contenu/lab practice/parts/submitted parts
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| - | The R0 riboregulator sequence is designed based on the | + | The R0 riboregulator sequence is designed based on the <a href="http://www.ncbi.nlm.nih.gov/pubmed/22454498">publication of Callura JM </a>. The principle is to introduce short regulatory sequences that create RNA secondary structures to better control protein expression. The basic design is reported in the following scheme (red hexagonal boxes, terminators; green oval box, rbs; blue and red rectangles, riboregulator sequences; arrows, promoters). Promoters P1 and P2 are controlling the expression of a single gene X (not present in the biobrick). Two terminators are placed between P1 and P2.<br> |
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<img src="https://static.igem.org/mediawiki/2013/9/9c/R0-1.png" class="imgcontent"/><br><br> | <img src="https://static.igem.org/mediawiki/2013/9/9c/R0-1.png" class="imgcontent"/><br><br> | ||
Revision as of 14:31, 25 September 2013
Submitted Parts
Gates
- BBa_K1132001: AND gate with recombinases switching gene regulatory sequences
- This AND gate was built with one promoter-terminator couple surrounded by the Bxb1 integrase sites and a second terminator surrounded by the Tp901.1 integrase sites. This system is designed to be activated only in the presence of both recombinases (transcription of the output gene). The switch is permanent.
The input signals for this gate are the production of either one or both integrases Bxb1 and Tp901.1. The output can be choosen at will by assembling this biobrick to any ORF containing an RBS site. We also designed a test Biobrick of the gate (BBa_K1132031) with the RFP protein as output.
This gate can be used in any regulation system, provided that the recombinases are assembled following the promoter of your choice with your specific regulations requirements. For example, if you want to activate the gate in presence of aTc and AHL, you just have to put the recombinase after the promoter activated by LuxR/AHL (BBa_R0065) and the promoter activated by aTc under the repression of TetR (BBa_R0040).
Even a relatively small amount of recombinases can switch the DNA fragments. Therefore, it is really important to control the recombinases expression with a well-locked promoter. You can look at our specially designed regulation sequence (riboregulator) to get as low as possible any undesired expression and production of the recombinases (BBa_K1132005, BBa_K1132006, BBa_K1132007, BBa_K1132008, BBa_K1132042).
In the present design, the strength of the promoter does not allow high level expression of the controlled output. However, change to stronger promoter than P7 should potentially lead to better expression levels.
Resetting the gate to its basal state requires a series of excisases capable of switching back the sequences to their native state.
The same type of design was used to build a XOR gate (BBa_K1132002).
- This AND gate was built with one promoter-terminator couple surrounded by the Bxb1 integrase sites and a second terminator surrounded by the Tp901.1 integrase sites. This system is designed to be activated only in the presence of both recombinases (transcription of the output gene). The switch is permanent.
- BBa_K1132002: XOR gate with recombinases switching gene regulatory sequences
- This XOR gate was built with one promoter- one terminator surrounded by the Bxb1 integrase sites and the Tp901.1 integrase sites. This system is designed to be activated only in the presence of excusively one recombinases (transcription of the output gene). The switch is permanent.
The input signals for this gate are the production of either one or both integrases Bxb1 and Tp901.1. The output can be choosen at will by assembling this biobrick to any ORF containing an RBS site. We also designed a test Biobrick of the gate (BBa_K1132032) with the RFP protein as output.
This gate can be used in any regulation system, provided that the recombinases are assembled following the promoter of your choice with your specific regulations requirements. For example, if you want to activate the gate in presence of aTc or AHL but not with both, you just have to put the recombinase after the promoter activated by LuxR/AHL (BBa_R0065) and the promoter activated by aTc under the repression of TetR (BBa_R0040).
Even a relatively small amount of recombinases can switch the DNA fragments. Therefore, it is really important to control the recombinases expression with a well-locked promoter. You can look at our specially designed regulation sequence (riboregulator) to get as low as possible any undesired expression and production of the recombinases (BBa_K1132005, BBa_K1132006, BBa_K1132007, BBa_K1132008, BBa_K1132042).
In the present design, the strength of the promoter does not allow high level expression of the controlled output. However, change to stronger promoter than P7 should potentially lead to better expression levels.
Resetting the gate to its basal state requires a series of excisases capable of switching back the sequences to their native state.
The same type of design was used to build a AND gate (BBa_K1132001).
Design of the gate :
For this gate, restrcition sites have been add between the promoter to change it esaly if necessary. Effectively, as shown during the caracterisation, the P7 promoter does not allow high level expression of the output. It could be interesting to change the promoter by a stronger one.
- This XOR gate was built with one promoter- one terminator surrounded by the Bxb1 integrase sites and the Tp901.1 integrase sites. This system is designed to be activated only in the presence of excusively one recombinases (transcription of the output gene). The switch is permanent.
- BBa_K1132003: AND gate with recombinases switching gene regulatory sequences and ORF
- This AND gate was built with one promoter surrounded by the PhiC31 integrase site and one coding sequence surrounded by the FimE integrase. To transcript a gene, a promoter has to be in front of the coding sequence, we need to have the promoter AND the coding sequence in the same way. It is an AND gate. In the basic state, the promoter and the gene are in the wrong way. The transcription of the output gene will occur in presence of the both promoter, when the promoter and the gene are in the right way.
The input signals for this gate are the production of either one or both integrases PhiC31 and FimE. The output can be choosen at will by insering between the recombinases sites of FimE any ORF containing an RBS site. To insert the reading fram, two restriction sites have been placed between the both FimE integrase sites, BamHI and ClaI. We also designed a test Biobrick of the gate (BBa_K1132034) with an inverted RFP protein as output.
This gate can be used in any regulation system, provided that the recombinases are assembled following the promoter of your choice with your specific regulations requirements. For example, if you want to activate the gate in presence of aTc and AHL, you just have to put the recombinase after the promoter activated by LuxR/AHL (BBa_R0065) and the promoter activated by aTc under the repression of TetR (BBa_R0040).
Even a relatively small amount of recombinases can switch the DNA fragments. Therefore, it is really important to control the recombinases expression with a well-locked promoter. You can look at our specially designed regulation sequence (riboregulator) to get as low as possible any undesired expression and production of the recombinases (BBa_K1132005, BBa_K1132006, BBa_K1132007, BBa_K1132008, BBa_K1132042).
In the present design, because of the promoter, the polymerase PolT7 is needed to express the gene. This promoter have been used, in order to have an higher level of expression, it can be assimlar to a amplificator. It is why we design one parts with the gate, the RFP inverted and the polymerase T7 after a promoter (BBa_K1132037).
Resetting the gate to its basal state requires a series of excisases capable of switching back the sequences to their native state.
The same type of design was used to build a XOR gate (BBa_K1132004).
Furthemore, if the gene is inserted inside the gate in the forward direction, the gate will not be an AND gate anymore, but it will only be activated in the presence of PhiC31 and in the absence of FimE.
- This AND gate was built with one promoter surrounded by the PhiC31 integrase site and one coding sequence surrounded by the FimE integrase. To transcript a gene, a promoter has to be in front of the coding sequence, we need to have the promoter AND the coding sequence in the same way. It is an AND gate. In the basic state, the promoter and the gene are in the wrong way. The transcription of the output gene will occur in presence of the both promoter, when the promoter and the gene are in the right way.
- BBa_K1132004: XOR gate with recombinases switching gene regulatory sequences and ORF
- This XOR gate was built with one promoter and one restriction sites BamHI surrounded by the PhiC31 integrase site and the FimE integrase. The idea is to insert an inverting gene between the recombinase sites with the help of the restriction site. To transcript the gene, the gene will need to be in the right way, to have been switched one time only by one of the both recombinases. This is a XOR gate.
The input signals for this gate are the production of either one or the both integrases PhiC31 and FimE. The output can be choosen at will by insering between the recombinases sites any ORF containing an RBS site. We also designed a test Biobrick of the gate (BBa_K1132035) with an inverted RFP protein as output.
This gate can be used in any regulation system, provided that the recombinases are assembled following the promoter of your choice with your specific regulations requirements. For example, if you want to activate the gate in presence of aTc and AHL, you just have to put the recombinase after the promoter activated by LuxR/AHL (BBa_R0065) and the promoter activated by aTc under the repression of TetR (BBa_R0040).
Even a relatively small amount of recombinases can switch the DNA fragments. Therefore, it is really important to control the recombinases expression with a well-locked promoter. You can look at our specially designed regulation sequence (riboregulator) to get as low as possible any undesired expression and production of the recombinases (BBa_K1132005, BBa_K1132006, BBa_K1132007, BBa_K1132008, BBa_K1132042).
In the present design, because of the promoter, the polymerase PolT7 is needed to express the gene. This promoter have been used, in order to have an higher level of expression, it can be assimlar to a amplificator. It is why we design one parts with the gate, the RFP inverted and the polymerase T7 after a promoter (BBa_K1132038).
Resetting the gate to its basal state requires a series of excisases capable of switching back the sequences to their native state.
In the same design, we build a XOR gate (BBa_K1132003).
Furthemore, if the gene is inserted inside the gate in the forward direction, the gate will not be an AND gate anymore, but it will only be activated in the presence of PhiC31 and in the absence of FimE.
- This XOR gate was built with one promoter and one restriction sites BamHI surrounded by the PhiC31 integrase site and the FimE integrase. The idea is to insert an inverting gene between the recombinase sites with the help of the restriction site. To transcript the gene, the gene will need to be in the right way, to have been switched one time only by one of the both recombinases. This is a XOR gate.
- BBa_K1132031: Test of the AND gate (BBa_K1132001) with RFP
- This BioBrick is design to test the AND gate (BBa_K1132001) by measuring the level of RFP after recombination events. The biobrick Bba_K081014 containing the RBS site, the coding sequence of the RFP and a terminator have been assembled with our gate.
- This BioBrick is design to test the AND gate (BBa_K1132001) by measuring the level of RFP after recombination events. The biobrick Bba_K081014 containing the RBS site, the coding sequence of the RFP and a terminator have been assembled with our gate.
- BBa_K1132032: Test of the XOR gate (BBa_K1132002) with RFP
- This BioBrick is design to test the XOR gate (BBa_K1132002) by measuring the level of RFP after recombination events. The biobrick Bba_K081014 containing the RBS site, the coding sequence of the RFP and a terminator have been assembled with our gate.
- This BioBrick is design to test the XOR gate (BBa_K1132002) by measuring the level of RFP after recombination events. The biobrick Bba_K081014 containing the RBS site, the coding sequence of the RFP and a terminator have been assembled with our gate.
- BBa_K1132034: AND gate (BBa_K1132003) with RFP
- This BioBrick is designed to test the AND gate (BBa_K1132003) by measuring the level of RFP after recombination events. The biobrick Bba_K081014 containing the RBS site, the coding sequence of the RFP has been inserted inside our gate between the FimE restrictions sites. However, to detect expression of RFP, the T7 polymerase is required. An amelioration of this BioBrick has been created, adding a strong promoter, an RBS and the open reading frame of the T7 polymerase to the construct (BBa_K1132037).
- This BioBrick is designed to test the AND gate (BBa_K1132003) by measuring the level of RFP after recombination events. The biobrick Bba_K081014 containing the RBS site, the coding sequence of the RFP has been inserted inside our gate between the FimE restrictions sites. However, to detect expression of RFP, the T7 polymerase is required. An amelioration of this BioBrick has been created, adding a strong promoter, an RBS and the open reading frame of the T7 polymerase to the construct (BBa_K1132037).
- BBa_K1132036: XOR gate (BBa_K1132004) with RFP
- This BioBrick is design to test the XOR gate (BBa_K1132004) by measuring the level of RFP after recombination events. The biobrick Bba_K081014 containing the RBS site, the coding sequence of the RFP and a terminator have been inserted inside our gate between the FimE restrictions sites.
- This BioBrick is design to test the XOR gate (BBa_K1132004) by measuring the level of RFP after recombination events. The biobrick Bba_K081014 containing the RBS site, the coding sequence of the RFP and a terminator have been inserted inside our gate between the FimE restrictions sites.
- BBa_K1132037: AND-inverted RFP gate (BBa_K1132034) with T7 polymerase under the control of a strong promoter, strong rbs
- This BioBrick is design to test the AND gate (BBa_K1132034) by measuring the level of RFP after recombination events. The biobrick Bba_K081014 containing the RBS site, the coding sequence of the RFP and a terminator have been inserted inside our gate between the FimE restrictions sites. The T7 polymerase gene is also present in the biobrick, under the control of a strong promoter, strong RBS. This part can therefore be used stand-alone as all elements to control the RFP output are present.
- This BioBrick is design to test the AND gate (BBa_K1132034) by measuring the level of RFP after recombination events. The biobrick Bba_K081014 containing the RBS site, the coding sequence of the RFP and a terminator have been inserted inside our gate between the FimE restrictions sites. The T7 polymerase gene is also present in the biobrick, under the control of a strong promoter, strong RBS. This part can therefore be used stand-alone as all elements to control the RFP output are present.
- BBa_K1132038: XOR-inverted RFP gate (BBa_K1132035) with T7 polymerase under the control of a strong promoter, strong rbs
- This BioBrick is design to test the XOR gate (BBa_K1132004) by measuring the level of RFP after recombination events. The biobrick Bba_K081014 containing the RBS site, the coding sequence of the RFP and a terminator have been inserted inside our gate between the FimE restrictions sites. The T7 polymerase gene is also present in the biobrick, under the control of a strong promoter, strong RBS. This part can therefore be used stand-alone as all elements to control the RFP output are present.
Riboregulators
- BBa_K1132005: R0 riboregulator switch with pTET and pLuxRCI
-
The R0 riboregulator sequence is designed based on the publication of Callura JM . The principle is to introduce short regulatory sequences that create RNA secondary structures to better control protein expression. The basic design is reported in the following scheme (red hexagonal boxes, terminators; green oval box, rbs; blue and red rectangles, riboregulator sequences; arrows, promoters). Promoters P1 and P2 are controlling the expression of a single gene X (not present in the biobrick). Two terminators are placed between P1 and P2.
Transcription occurring at P2 promoter stops at terminators placed after gene X. However, the red sequence can fold into a RNA secondary structure, blocking the RBS, preventing ribosome binding. The gene X immediately placed after the RBS is transcribed but not translated (no protein X).
The P1 promoter controls the expression of a small riboregulatory sequence capable of interacting with the one blocking RBS, but stronger in term of interaction. If transcription occurs at both sites P1 and P2, a small RNA is produced that destabilizes the loop created on the RBS and therefore releasing the riboregulator and enabling translation.
The part is released without any gene and can therefore be used to better control any protein expression. The P1 promoter is pTET (BBa_R0040) and the P2 promoter is pLuxR (BBA_R0065). pLuxR can be changed with Bam HI and Cla I restriction sites.
-
The R0 riboregulator sequence is designed based on the publication of Callura JM . The principle is to introduce short regulatory sequences that create RNA secondary structures to better control protein expression. The basic design is reported in the following scheme (red hexagonal boxes, terminators; green oval box, rbs; blue and red rectangles, riboregulator sequences; arrows, promoters). Promoters P1 and P2 are controlling the expression of a single gene X (not present in the biobrick). Two terminators are placed between P1 and P2.
- BBa_K1132001: AND gate with recombinases switching gene regulatory sequences
- This AND gate was built with one promoter-terminator couple surrounded by the Bxb1 integrase sites and a second terminator surrounded by the Tp901.1 integrase sites. This system is designed to be activated only in the presence of both recombinases (transcription of the output gene). The switch is permanent.
The input signals for this gate are the production of either one or both integrases Bxb1 and Tp901.1. The output can be choosen at will by assembling this biobrick to any ORF containing an RBS site. We also designed a test Biobrick of the gate (BBa_K1132031) with the RFP protein as output.
This gate can be used in any regulation system, provided that the recombinases are assembled following the promoter of your choice with your specific regulations requirements. For example, if you want to activate the gate in presence of aTc and AHL, you just have to put the recombinase after the promoter activated by LuxR/AHL (BBa_R0065) and the promoter activated by aTc under the repression of TetR (BBa_R0040).
Even a relatively small amount of recombinases can switch the DNA fragments. Therefore, it is really important to control the recombinases expression with a well-locked promoter. You can look at our specially designed regulation sequence (riboregulator) to get as low as possible any undesired expression and production of the recombinases (BBa_K1132005, BBa_K1132006, BBa_K1132007, BBa_K1132008, BBa_K1132042).
In the present design, the strength of the promoter does not allow high level expression of the controlled output. However, change to stronger promoter than P7 should potentially lead to better expression levels.
Resetting the gate to its basal state requires a series of excisases capable of switching back the sequences to their native state.
The same type of design was used to build a XOR gate (BBa_K1132002).
- This AND gate was built with one promoter-terminator couple surrounded by the Bxb1 integrase sites and a second terminator surrounded by the Tp901.1 integrase sites. This system is designed to be activated only in the presence of both recombinases (transcription of the output gene). The switch is permanent.
Recombinases
- BBa_K1132000: T7 RNA Polymerase
- Coding sequence of T7 RNA polymerase. It permits transcription of DNA under control of promoter T7. This part does not include any promoter, rbs or terminator. It was extracted from B21-DE3 genome by PCR reaction.
- BBa_K1132001: AND gate with recombinases switching gene regulatory sequences
- This AND gate was built with one promoter-terminator couple surrounded by the Bxb1 integrase sites and a second terminator surrounded by the Tp901.1 integrase sites. This system is designed to be activated only in the presence of both recombinases (transcription of the output gene). The switch is permanent.
The input signals for this gate are the production of either one or both integrases Bxb1 and Tp901.1. The output can be choosen at will by assembling this biobrick to any ORF containing an RBS site. We also designed a test Biobrick of the gate (BBa_K1132031) with the RFP protein as output.
This gate can be used in any regulation system, provided that the recombinases are assembled following the promoter of your choice with your specific regulations requirements. For example, if you want to activate the gate in presence of aTc and AHL, you just have to put the recombinase after the promoter activated by LuxR/AHL (BBa_R0065) and the promoter activated by aTc under the repression of TetR (BBa_R0040).
Even a relatively small amount of recombinases can switch the DNA fragments. Therefore, it is really important to control the recombinases expression with a well-locked promoter. You can look at our specially designed regulation sequence (riboregulator) to get as low as possible any undesired expression and production of the recombinases (BBa_K1132005, BBa_K1132006, BBa_K1132007, BBa_K1132008, BBa_K1132042).
In the present design, the strength of the promoter does not allow high level expression of the controlled output. However, change to stronger promoter than P7 should potentially lead to better expression levels.
Resetting the gate to its basal state requires a series of excisases capable of switching back the sequences to their native state.
The same type of design was used to build a XOR gate (BBa_K1132002).
- This AND gate was built with one promoter-terminator couple surrounded by the Bxb1 integrase sites and a second terminator surrounded by the Tp901.1 integrase sites. This system is designed to be activated only in the presence of both recombinases (transcription of the output gene). The switch is permanent.
Input signals
- BBa_K1132000: T7 RNA Polymerase
- Coding sequence of T7 RNA polymerase. It permits transcription of DNA under control of promoter T7. This part does not include any promoter, rbs or terminator. It was extracted from B21-DE3 genome by PCR reaction.
- BBa_K1132001: AND gate with recombinases switching gene regulatory sequences
- This AND gate was built with one promoter-terminator couple surrounded by the Bxb1 integrase sites and a second terminator surrounded by the Tp901.1 integrase sites. This system is designed to be activated only in the presence of both recombinases (transcription of the output gene). The switch is permanent.
The input signals for this gate are the production of either one or both integrases Bxb1 and Tp901.1. The output can be choosen at will by assembling this biobrick to any ORF containing an RBS site. We also designed a test Biobrick of the gate (BBa_K1132031) with the RFP protein as output.
This gate can be used in any regulation system, provided that the recombinases are assembled following the promoter of your choice with your specific regulations requirements. For example, if you want to activate the gate in presence of aTc and AHL, you just have to put the recombinase after the promoter activated by LuxR/AHL (BBa_R0065) and the promoter activated by aTc under the repression of TetR (BBa_R0040).
Even a relatively small amount of recombinases can switch the DNA fragments. Therefore, it is really important to control the recombinases expression with a well-locked promoter. You can look at our specially designed regulation sequence (riboregulator) to get as low as possible any undesired expression and production of the recombinases (BBa_K1132005, BBa_K1132006, BBa_K1132007, BBa_K1132008, BBa_K1132042).
In the present design, the strength of the promoter does not allow high level expression of the controlled output. However, change to stronger promoter than P7 should potentially lead to better expression levels.
Resetting the gate to its basal state requires a series of excisases capable of switching back the sequences to their native state.
The same type of design was used to build a XOR gate (BBa_K1132002).
- This AND gate was built with one promoter-terminator couple surrounded by the Bxb1 integrase sites and a second terminator surrounded by the Tp901.1 integrase sites. This system is designed to be activated only in the presence of both recombinases (transcription of the output gene). The switch is permanent.
General Inducer
- BBa_K1132000: T7 RNA Polymerase
- Coding sequence of T7 RNA polymerase. It permits transcription of DNA under control of promoter T7. This part does not include any promoter, rbs or terminator. It was extracted from B21-DE3 genome by PCR reaction.
- BBa_K1132001: AND gate with recombinases switching gene regulatory sequences
- This AND gate was built with one promoter-terminator couple surrounded by the Bxb1 integrase sites and a second terminator surrounded by the Tp901.1 integrase sites. This system is designed to be activated only in the presence of both recombinases (transcription of the output gene). The switch is permanent.
The input signals for this gate are the production of either one or both integrases Bxb1 and Tp901.1. The output can be choosen at will by assembling this biobrick to any ORF containing an RBS site. We also designed a test Biobrick of the gate (BBa_K1132031) with the RFP protein as output.
This gate can be used in any regulation system, provided that the recombinases are assembled following the promoter of your choice with your specific regulations requirements. For example, if you want to activate the gate in presence of aTc and AHL, you just have to put the recombinase after the promoter activated by LuxR/AHL (BBa_R0065) and the promoter activated by aTc under the repression of TetR (BBa_R0040).
Even a relatively small amount of recombinases can switch the DNA fragments. Therefore, it is really important to control the recombinases expression with a well-locked promoter. You can look at our specially designed regulation sequence (riboregulator) to get as low as possible any undesired expression and production of the recombinases (BBa_K1132005, BBa_K1132006, BBa_K1132007, BBa_K1132008, BBa_K1132042).
In the present design, the strength of the promoter does not allow high level expression of the controlled output. However, change to stronger promoter than P7 should potentially lead to better expression levels.
Resetting the gate to its basal state requires a series of excisases capable of switching back the sequences to their native state.
The same type of design was used to build a XOR gate (BBa_K1132002).
- This AND gate was built with one promoter-terminator couple surrounded by the Bxb1 integrase sites and a second terminator surrounded by the Tp901.1 integrase sites. This system is designed to be activated only in the presence of both recombinases (transcription of the output gene). The switch is permanent.
T7
- BBa_K1132000: T7 RNA Polymerase
- Coding sequence of T7 RNA polymerase. It permits transcription of DNA under control of promoter T7. This part does not include any promoter, rbs or terminator. It was extracted from B21-DE3 genome by PCR reaction.
- BBa_K1132044: Strong promoter-strong RBS-T7 RNA polymerase
- Coding sequence of T7 RNA polymerase under control of strong promoter. It also include strong rbs.
- BBa_K1132045: Promoter T7-RFP
- Coding sequence of RFP ( BBa_K081014) under control of T7 promoter (BBa_I712074). T7 promoter is very specific promoter which is transcribed only by specific T7 RNA polymerase. Then RFP will be produced only in the presence of T7 RNA Polymerase ( BBa_K1132000). The RFP coding sequence is flanked by a rbs and a terminator. Red color can be seen after 18 hours. Such a system permits strong and specific transcription of RFP.
