Difference between revisions of "Part:BBa K5439004"
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| − | + | =Usage and Biology= | |
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| − | + | = Characterization = | |
| − | + | == Gene Amplification, Assembly and Transformation == | |
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| − | The basis for the assembly of this composite part was the previous iteration of the biosensor, ECFP_EryK_mVenus[https://parts.igem.org/Part:BBa K4447004 (BBa K4447004)], which was made up of the 3 genes in a pET-28b backbone. In order to successfully assemble the construct through Gibson Assembly without scars between parts and ensure proper expression of the full fusion construct, we amplified the vector using primers that bind to the ends of both fluorescent proteins and exclude the center EryK gene, obtaining an empty FRET backbone with the homology regions corresponding to the gene of interest, in this case TjPCs. This was the basis for the construction of the other two versions of the biosensor: ECFP_RifMo_mVenus [https://parts.igem.org/Part: | + | The basis for the assembly of this composite part was the previous iteration of the biosensor, ECFP_EryK_mVenus[https://parts.igem.org/Part:BBa K4447004 (BBa K4447004)], which was made up of the 3 genes in a pET-28b backbone. In order to successfully assemble the construct through Gibson Assembly without scars between parts and ensure proper expression of the full fusion construct, we amplified the vector using primers that bind to the ends of both fluorescent proteins and exclude the center EryK gene, obtaining an empty FRET backbone with the homology regions corresponding to the gene of interest, in this case TjPCs. This was the basis for the construction of the other two versions of the biosensor: ECFP_RifMo_mVenus [https://parts.igem.org/Part:BBa_K5439003 (BBa_K5439003)], and ECFP_IpfF_mVenus [https://parts.igem.org/Part:BBa_K5439006 (BBa_K5439006)]. This method of assembly effectively makes our system modular and customizable, as the detector gene can be switched out to cover a wider range of contaminants. |
| + | Along with amplifying the FRET backbone, we amplified TjPCs [https://parts.igem.org/Part:BBa_K5439001 (BBa_K5439001)]with primers that generate homology regions corresponding to those generated by the backbone amplification. After several optimization cycles, which included optimizing the annealing temperature, number of cycles, and elongation times, we obtained purified fragments to use in the assembly. <b> Figure 2 </b> displays the PCR gels for both the vector and the gene. | ||
| − | === Protein expression and fluorescence validation | + | <html> |
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| + | <img src="https://static.igem.wiki/teams/5439/amplificacion-pcr.png" width="500"> | ||
| + | <figcaption><b>Figure 2.</b> (A) Agarose gel showing the amplified TjPCs gene used for the assembly of the construct. The band corresponds to the approximate length of the TjPCs gene (~1455 bp) (B) Agarose gel showing the amplification of the FRET backbone in pET28b. The faint band corresponds to the amplicon's expected length (~6800 bp), while the more visible band corresponds to unamplified plasmid in its supercoiled form. </figcaption> | ||
| + | </figure> | ||
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| + | == Protein expression and fluorescence validation == | ||
| − | + | = References = | |
Revision as of 04:52, 2 October 2024
FRET-based system for the detection of cadmium
FRET-based sensor system for the detection of cadmium and other heavy metals that consists of phytochelatin synthase from Thlaspi japnonicum (BBa_K5439001),an enzyme that catalyzes the biosynthesis of phytochelatins using as a co-substrate the heavy metal cadmium, flanked by two fluorescent proteins: ECFP (BBa_K1159302)as an energy donor and mVenus (BBa_K1907000)as an energy acceptor.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 895
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 745
Illegal BglII site found at 2154
Illegal XhoI site found at 2176 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2825
Contents
Usage and Biology
Characterization
Gene Amplification, Assembly and Transformation
The basis for the assembly of this composite part was the previous iteration of the biosensor, ECFP_EryK_mVenusK4447004 (BBa K4447004), which was made up of the 3 genes in a pET-28b backbone. In order to successfully assemble the construct through Gibson Assembly without scars between parts and ensure proper expression of the full fusion construct, we amplified the vector using primers that bind to the ends of both fluorescent proteins and exclude the center EryK gene, obtaining an empty FRET backbone with the homology regions corresponding to the gene of interest, in this case TjPCs. This was the basis for the construction of the other two versions of the biosensor: ECFP_RifMo_mVenus (BBa_K5439003), and ECFP_IpfF_mVenus (BBa_K5439006). This method of assembly effectively makes our system modular and customizable, as the detector gene can be switched out to cover a wider range of contaminants.
Along with amplifying the FRET backbone, we amplified TjPCs (BBa_K5439001)with primers that generate homology regions corresponding to those generated by the backbone amplification. After several optimization cycles, which included optimizing the annealing temperature, number of cycles, and elongation times, we obtained purified fragments to use in the assembly. Figure 2 displays the PCR gels for both the vector and the gene.
