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).

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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.

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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 sites and one coding sequence surrounded by the FimE sites. 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 recombinases, 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 recombinases 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 recombinases 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.

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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 sites. 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 recombinases 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_K1132036) 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.

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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.



On the Registry

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.



On the Registry

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).



On the Registry

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.



On the Registry

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.

On the Registry

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.

On the Registry

BBa_K1132005: R0 riboregulator switch with pTET and pLuxRCI

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.



On the Registry

BBa_K1132006: R1 riboregulator switch with with pTET and pLuxRCI

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 pLuxRCI (BBA_R0065). pLuxRCI can be changed with Bam HI and Cla I restriction sites.



On the Registry

BBa_K1132007: R2 riboregulator switch with with pTET and pFixJ

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 pFixJ (Bba_K592006). pFixJ can be changed with Bam HI and Cla I restriction sites.



On the Registry

BBa_K1132008: R4 riboregulator switch with with pTET and pOmpC

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 pOmpC (BBA_R0082). P2 can be changed with Bam HI and Cla I restriction sites.



On the Registry

BBa_K1132042: R1-pLac riboregulator switch with with pTET and pLac

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 pLac (BBA_R0011). P2 can be changed with Bam HI and Cla I restriction sites.



On the Registry

BBa_K1132043: R1-pLac-RFP

Assembly between BBa_K1132042 and BBa_E1010. On the Registry

BBa_K1132046: R1-pLac-Bxb1

Assembly between BBa_K1132042 and BBa_K907000. On the Registry

BBa_K1132025: FimE integrase coding sequence with constitutive weak promoter and terminator

FimE integrase allows DNA 180° switching between two recognition sites (IRL: BBa_K137010 and IRR: BBa_K137008). As the protein is very active even at low level of expression, it is associated with a constitutive weak promoter.
On the Registry

BBa_K1132026: FimE integrase coding sequence with RBS and terminator

FimE integrase allows DNA 180° switching between two recognition sites (IRL: BBa_K137010 and IRR: BBa_K137008).
On the Registry

BBa_K1132027: Bxb1 integrase coding sequence with constitutive weak promoter

Bxb1 integrase allows DNA 180° switching between two recognition sites (attB and attP). As the protein is very active even at low level of expression, it is associated with a constitutive weak promoter. See BBa_K907000 for further information.
On the Registry

BBa_K1132028: Bxb1 integrase coding sequence with RBS

Bxb1 integrase allows DNA 180° switching between two recognition sites (attB and attP). See BBa_K907000 for further infomation.
On the Registry

BBa_K1132011: heme oxygenase (ho1) and ferredoxin oxidoreductase (PcyA)

Two required genes for phycocyanobilin (PCB) biosynthesis.
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.
On the Registry

BBa_K1132012: heme oxygenase (ho1), ferredoxin oxidoreductase (PcyA) and tetracycline repressor (TetR)

Two genes required for phycocyanobilin (PCB) biosynthesis and the coding region for the TetR protein.

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.

On the Registry

BBa_K1132013: heme oxygenase (ho1), ferredoxin oxidoreductase (PcyA) and tetracycline repressor (TetR) under constitutive promoter

Two genes required for phycocyanobilin (PCB) biosynthesis and the coding region for the TetR protein with Ribosome Binding Site (RBS) under a strong promoter. The Part:BBa_K1132012 was inserted behind BBa_K608003, a strong promoter with a medium RBS.

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.

On the Registry

BBa_K1132014: Promoter (OmpR, positive) followed by RFP protein generator

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 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.

On the Registry

BBa_K1132015: Cph8 (Cph1/EnvZ fusion) under TetR repressible promoter

Chimeric Cph1 light receptor/EnvZ protein requires phycocyanobilin (PCB) biosynthetic genes for PCB formation (ho1 and PcyA). The exposure to red light (660nm) inhibits the activity of the EnvZ histidine kinase domain. In the dark, the EnvZ histidine kinase phosphorylates endogenous OmpR, a transcription factor which activates transcription from the OmpC promoter. This part must be used in E.coli deficient in wild-type EnvZ.

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.
On the Registry

BBa_K1132016: Promoter (OmpR, positive) followed by RFP protein generator and Cph8 (Cph1/EnvZ fusion) under TetR repressible promoter

Chimeric Cph1 light receptor/EnvZ protein requires phycocyanobilin (PCB) biosynthetic genes for PCB formation (ho1 and PcyA). The exposure to red light (660nm) inhibits the activity of the EnvZ histidine kinase domain. In the dark, the EnvZ histidine kinase phosphorylates endogenous OmpR, a transcription factor which activates transcription from the OmpC promoter. This part must be used in E.coli deficient in wild-type EnvZ.

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.

On the Registry

BBa_K1132017: For the characterisation of the red light responsive system, under tetR regulation

Chimeric Cph1 light receptor/EnvZ (Cph8) protein requires phycocyanobilin (PCB) biosynthetic genes for PCB formation (ho1 and PcyA). The exposure to red light (660nm) inhibits the activity of the EnvZ histidine kinase domain. In the dark, the EnvZ histidine kinase phosphorylates endogenous OmpR, a transcription factor which activates transcription from the OmpC promoter. This part must be used in E.coli deficient in wild-type EnvZ.

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.
On the Registry

BBa_K1132018: FixK2 promoter-rbs-mRFP-term

This biobrick was created by assembling two preexisting biobricks: a gene encoding a mutated version of the red fluorescent protein (BBa_K081014) with rbs and terminator and put under the control of FixK2 (BBa_K592006), a blue light sensitive promoter (465-482nm). FixK2 was previously described by iGEM11_Uppsala-Sweden as working with FixJ.
When FixJ is phosphorylated, it can bind FixK2 and thus activate the downstream system.
In the litterature, FixJ is phosphorylated by YF1 in darkness and unphosphorylated in light. BBa_K592016 is a composite part using YF1 with FixJ.


On the Registry

BBa_K1132019: term-FixK2 promoter-rbs-mRFP1-term

A terminator was assembled to BBa_K1132018 (FixK2 promoter-mRFP1). This is an intermediate construction priorr the final YF1-FixJ biobrick.
On the Registry

BBa_K1132020: rbs-YF1-rbs-FixJ-term-pFixK2-mRFP1

This composite part can be used for a blue light sensitive system. It is built with 4 preexisting parts: rbs-YF1-rbs-FixJ (BBa_K592016) with a terminator (BBa_B1010) and followed by FixK2 (BBa_K081014)-rbs-mRFP1-term (BBa_K592006). Rbs-YF1-rbs-FixJ is a regulatory protein working with the promotor FixK2. In darkness, YF1 is phosphorylated and gives its phosphate group to FixJ. Once phosphorylated, FixJ can bind to FixK2 promoter and actives it. In light, FixK2 promoter can't work.
A final construction was made with a constitutive promoter (Bba_J23116) but this promoter seems to weak to generate enought copies of YF1-FixJ to activate FixK2.
Adding a strong promoter before this biobrick can improve the system.
On the Registry

BBa_K1132021: TetR-pTet-rbs-mRFP1-term

This biobrick is a tool to use the couple TetR-pTet system. In this construct, a modified coding region of TetR (BBa_C0040) was assembled before pTet-mRFP (Bba_I13521). pTet is a constitutive promoter. TetR is an inducer that binds to pTet promoter and thus, represses the expression of the downstream gene controled by pTet. Supply of tetracycline or its analog aTc (anhydrotetracycline) is known to bind to tetR and invert the operation (inhibits expression of mRFP in this case). A further construct was made by assembling the promoter BBa_J23116 upstream. (Construct available here: BBa_K1132022).
On the Registry

BBa_K1132022: BBa_J23116-TetR-pTet-RFP

The couple TetR-pTet system was already described in the litterature. This construction is a first step to characterize the entire system. A constitutive promoter (BBa_J23116) was assemble to tetR and then assemble to pTet-rbs-RFP-term. pTet is a constitutive promoter. TetR is an inducer that binds to pTet promoter and thus, represses expression the downstream system. Supply of tetracycline or its analog aTc (anhydrotetracycline) is known to bind to tetR and invert the operation (inhibits expression of mRFP in this case).

After 18 hours, clones containing the plasmid (BBa_J23116-TetR-pTet-RFP) show a leaky basal expression of mRFP. We suppose that the promoter was too weak to express TetR in large quantity. Assembly of a stronger promoter could improve the system and lock the response to an ON/OFF response.

Besides, an experience was done to analyze the effect of the aTc inducer. Result show a visible induction of the red fluorescent protein expression by addition of aTC (60 ng/mL).




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On the Registry

BBa_K1132023: strong prom-strong rbs-rbs-tetR-term

A strong promoter with strong rbs was assembled to a preexisting biobrick rbs-TetR-term ( BBa_P0440) and permits to express TetR in great quantity. The presence of two ribosome binding sites before TetR coding sequence doesn't show any issue to express TetR. The system of TetR-pTet was described there: BBa_K1132022

On the Registry

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.

On the Registry

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.

On the Registry

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.

On the Registry