Team:INSA Toulouse/contenu/lab practice/parts/submitted parts
From 2013.igem.org
Submitted Parts
Gates
BBa_K1132001: AND gate with recombinases switching gene regulatory sequences
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 () 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 ([http://parts.igem.org/Part:BBa_R0040 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 ([http://parts.igem.org/Part:BBa_K1132005 BBa_K1132005], [http://parts.igem.org/Part:BBa_K1132006 BBa_K1132006], [http://parts.igem.org/Part:BBa_K1132007 BBa_K1132007], [http://parts.igem.org/Part:BBa_K11320308 BBa_K1132008], [http://parts.igem.org/Part:BBa_K1132042 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 ([http://parts.igem.org/Part:BBa_K1132002 BBa_K1132002]). 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).
BBa_K1132002: XOR gate with recombinases switching gene regulatory sequences
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.
BBa_K1132003: AND gate with recombinases switching gene regulatory sequences and ORF
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.
BBa_K1132004: XOR gate with recombinases switching gene regulatory sequences and ORF
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.
BBa_K1132031: Test of the AND gate (BBa_K1132001) with RFP
BBa_K1132032: Test of the XOR gate (BBa_K1132002) with RFP
BBa_K1132034: AND gate (BBa_K1132003) with RFP
BBa_K1132036: XOR gate (BBa_K1132004) with RFP
BBa_K1132037: AND-inverted RFP gate (BBa_K1132034) with T7 polymerase under the control of a strong promoter, strong rbs
BBa_K1132038: XOR-inverted RFP gate (BBa_K1132035) with T7 polymerase under the control of a strong promoter, strong rbs
Riboregulators
How it works?
These riboregulators sequences are 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.
BBa_K1132005: R0 riboregulator switch with pTET and pLuxRCI
BBa_K1132006: R1 riboregulator switch with with pTET and pLuxRCI
BBa_K1132007: R2 riboregulator switch with with pTET and pFixJ
BBa_K1132008: R4 riboregulator switch with with pTET and pOmpC
BBa_K1132042: R1-pLac riboregulator switch with with pTET and pLac
BBa_K1132043: R1-pLac-RFP
BBa_K1132046: R1-pLac-Bxb1
Recombinases
BBa_K1132025: FimE integrase coding sequence with constitutive weak promoter and terminator
BBa_K1132026: FimE integrase coding sequence with RBS and terminator
BBa_K1132027: Bxb1 integrase coding sequence with constitutive weak promoter
BBa_K1132028: Bxb1 integrase coding sequence with RBS
BBa_K1132010: Tp901 integrase
The 180° switch permits to design a lot of regulation tools, such as logical gates that can be found here (BBa_K1132001, BBa_K1132002).
Input signals
BBa_K1132011: heme oxygenase (ho1) and ferredoxin oxidoreductase (PcyA)
ho1 produces biliverdin IXalpha, it represents the first of two steps in PCB biosynthesis. PcyA converts biliverdin IXalpha to PCB. PCB associates with EnvZ fusion protein, Cph8 (Part:BBa_I15010), to transduce a light signal into a genetic response.
BBa_K1132012: heme oxygenase (ho1), ferredoxin oxidoreductase (PcyA) and tetracycline repressor (TetR)
ho1 produces biliverdin IXalpha, it represents the first of two steps in PCB biosynthesis. PcyA converts biliverdin IXalpha to PCB. PCB associates with EnvZ fusion protein, Cph8 (Part:BBa_I15010), to transduce a light signal into a genetic response.
TetR binds to the p(TetR) (Part:BBa_R0040) and inhibits its operation. It regulates the light sensor Cph8 (Part:BBa_K1132015). aTc (anhydrotetracycline) binds to TetR and allows Cph8 production.
BBa_K1132013: heme oxygenase (ho1), ferredoxin oxidoreductase (PcyA) and tetracycline repressor (TetR) under constitutive promoter
ho1 produces biliverdin IXalpha, it represents the first of two steps in PCB biosynthesis. PcyA converts biliverdin IXalpha to PCB. PCB associates with EnvZ fusion protein, Cph8 (Part:BBa_I15010), to transduce a light signal into a genetic response.
TetR binds to the p(TetR) (Part:BBa_R0040) and inhibits its operation. It regulates the light sensor Cph8 (Part:BBa_K1132015). aTc (anhydrotetracycline) binds to TetR and allows Cph8 production. Both of these two parts construct the red light responsive system, under tetR regulation.
BBa_K1132014: Promoter (OmpR, positive) followed by RFP protein generator
This is a test Biobrick for the Red light responsive system (Part:BBa_K1132013 and Part:BBa_K1132015) with the RFP protein as output. EnvZ phosphorylates OmpR to OmpR-P that activate the promoter. It must be used in E.coli deficient in wild-type EnvZ.
BBa_K1132015: Cph8 (Cph1/EnvZ fusion) under TetR repressible promoter
The promoter p(TetR) is constitutively on and repressed by TetR (Part:BBa_P0440). TetR binds to the p(TetR) and inhibits its operation. It regulates the light sensor Cph8. Tetracycline or aTc (anhydrotetracycline) binds to TetR and allows Cph8 production.
This part can be used with Part:BBa_K1132013 composed of the heme oxygenase (ho1), ferredoxin oxidoreductase (PcyA) and tetracycline repressor (TetR) under constitutive promoter. Both of these parts form a red light responsive system, under tetR regulation.
BBa_K1132016: Promoter (OmpR, positive) followed by RFP protein generator and Cph8 (Cph1/EnvZ fusion) under TetR repressible promoter
The promoter p(TetR) is constitutively on and repressed by TetR (Part:BBa_P0440). TetR binds to the p(TetR) and inhibits its operation. It regulates Cph8 (Part:BBa_K1132015) production. Tetracycline or aTc (anhydrotetracycline) binds to TetR and allows Cph8 production.
This part is an intermediate Biobrick for the characterisation of the red light responsive system (Part:BBa_K1132017) with the RFP protein as output. The monomeric RFP is a Red Fluorescent Protein with an excitation peak at 584 nm and an emission peak at 607 nm. The RFP is positively regulated by OmpR-controlled promoter. Phosphorylated OmpR binds to the operator sites and activates transcription of RFP.
This must be used with Part:BBa_K1132013 composed of the heme oxygenase (ho1), ferredoxin oxidoreductase (PcyA) and tetracycline repressor (TetR) under constitutive promoter.
BBa_K1132017: For the characterisation of the red light responsive system, under tetR regulation
The heme oxygenase (ho1), ferredoxin oxidoreductase (PcyA) and tetracycline repressor (TetR) are under a constitutive promoter. Ho1 and PcyA are genes required for phycocyanobilin (PCB) biosynthesis and TetR binds to the p(TetR) (Part:BBa_R0040) that regulated light sensor (Cph8). aTc (anhydrotetracycline) binds to TetR and allows Cph8 production.
The RFP is positively regulated by OmpR-controlled promoter. Phosphorylated OmpR binds to the operator sites and activates transcription of RFP. This is a test Biobrick for the red light responsive system with the RFP protein as output.
BBa_K1132018: pFixK2-RFP
BBa_K1132019: t-pFixK2-RFP
BBa_K1132020: YF1-FixJ-t-pFixK2-RFP
General Inducer
BBa_K1132021: tetR-ptet-RFP
BBa_K1132022: Pfaible-tetR-pTet-RFP
BBa_K1132023: strong promoter - strong rbs - tetR
T7
BBa_K1132000: T7 RNA Polymerase
BBa_K1132044: Strong promoter-strong RBS-T7 RNA polymerase
BBa_K1132045: Promoter T7-RFP