Difference between revisions of "Part:BBa K2970006"
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This gate is a toehold switch system with which a gene of interest can be locked and regulated on a translational level using mRNA as regulator. After transcription, the mRNA of this gate forms a hairpin that hides the ribosome binding site and start codon of the gene of interest, thus translation can not occur. A complementary part to the gate (trigger) is needed to open the hairpin and release the ribosome binding site. In this case two triggers are needed that form a trigger complex to open the gate (BBa_K2970000 and BBa_K2970001). The affinity between the trigger complex and the gate is greater than that of the gate to itself (in the loop). A single trigger cannot open the gate because it has only half the required complementary sequence. | This gate is a toehold switch system with which a gene of interest can be locked and regulated on a translational level using mRNA as regulator. After transcription, the mRNA of this gate forms a hairpin that hides the ribosome binding site and start codon of the gene of interest, thus translation can not occur. A complementary part to the gate (trigger) is needed to open the hairpin and release the ribosome binding site. In this case two triggers are needed that form a trigger complex to open the gate (BBa_K2970000 and BBa_K2970001). The affinity between the trigger complex and the gate is greater than that of the gate to itself (in the loop). A single trigger cannot open the gate because it has only half the required complementary sequence. | ||
| − | To | + | To use this system in bacteria we implemented the gate sequence together with a gene for chloramphenicol (<partinfo>BBa_K2970011</partinfo>), flanked by a constitutive promoter (<partinfo>BBa_J23100</partinfo>) and a strong terminator (<partinfo>BBa_B1002</partinfo>) into <partinfo>pSB1A3</partinfo> where the ampicillin resistance can be cut out. |
After transformation of both trigger plasmids (<partinfo>BBa_K2970003</partinfo> and <partinfo>BBa_K2970004</partinfo>) and the gate plasmid in one bacterium all three mRNA structures will be formed, the gate will be opened, and the translation of the chloramphenicol resistance can take place. Bacteria that took all three plasmids are able to survive on media with chloramphenicol. This system can be used to transform many genes of interest on three different plasmids into bacteria with only using one antibiotic resistance instead of three different resistances. | After transformation of both trigger plasmids (<partinfo>BBa_K2970003</partinfo> and <partinfo>BBa_K2970004</partinfo>) and the gate plasmid in one bacterium all three mRNA structures will be formed, the gate will be opened, and the translation of the chloramphenicol resistance can take place. Bacteria that took all three plasmids are able to survive on media with chloramphenicol. This system can be used to transform many genes of interest on three different plasmids into bacteria with only using one antibiotic resistance instead of three different resistances. | ||
Revision as of 12:26, 20 October 2019
Gate Composition
This gate is a toehold switch system with which a gene of interest can be locked and regulated on a translational level using mRNA as regulator. After transcription, the mRNA of this gate forms a hairpin that hides the ribosome binding site and start codon of the gene of interest, thus translation can not occur. A complementary part to the gate (trigger) is needed to open the hairpin and release the ribosome binding site. In this case two triggers are needed that form a trigger complex to open the gate (BBa_K2970000 and BBa_K2970001). The affinity between the trigger complex and the gate is greater than that of the gate to itself (in the loop). A single trigger cannot open the gate because it has only half the required complementary sequence.
To use this system in bacteria we implemented the gate sequence together with a gene for chloramphenicol (BBa_K2970011), flanked by a constitutive promoter (BBa_J23100) and a strong terminator (BBa_B1002) into pSB1A3 where the ampicillin resistance can be cut out.
After transformation of both trigger plasmids (BBa_K2970003 and BBa_K2970004) and the gate plasmid in one bacterium all three mRNA structures will be formed, the gate will be opened, and the translation of the chloramphenicol resistance can take place. Bacteria that took all three plasmids are able to survive on media with chloramphenicol. This system can be used to transform many genes of interest on three different plasmids into bacteria with only using one antibiotic resistance instead of three different resistances.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]