Submitted Parts

Basic Parts

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).

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.

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.

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.

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