Difference between revisions of "Part:BBa K4447004"
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| − | + | With the rise of synthetic biology, biosensors have gained popularity over the years. Biosensors are devices that detect the presence of certain compounds by using living organisms or biological molecules. | |
In this composite part, we propose an enzymatic system based on how the Förster resonance energy transfer (FRET) operates: one enzyme capable of recognizing and degrading erythromycin will be flanked by two fluorescent proteins. This part incorporates NcoI and XhoI restriction sites in 5' and 3' ends for protein overexpression in pBAD/Myc-His plasmids, a gly-gly-ser spacer, and a polyhistidine tag before stop codon at the end of Venus for protein purification. Any linker does not separate each protein; for instance, stop codons for ECFP and erythromycin C-12 hydroxylase were removed. <b>Figure 1</b> displays the three-dimensional structure of this protein system. | In this composite part, we propose an enzymatic system based on how the Förster resonance energy transfer (FRET) operates: one enzyme capable of recognizing and degrading erythromycin will be flanked by two fluorescent proteins. This part incorporates NcoI and XhoI restriction sites in 5' and 3' ends for protein overexpression in pBAD/Myc-His plasmids, a gly-gly-ser spacer, and a polyhistidine tag before stop codon at the end of Venus for protein purification. Any linker does not separate each protein; for instance, stop codons for ECFP and erythromycin C-12 hydroxylase were removed. <b>Figure 1</b> displays the three-dimensional structure of this protein system. | ||
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=Selecting Fluorescent Proteins= | =Selecting Fluorescent Proteins= | ||
Revision as of 22:09, 29 September 2022
FRET-based system for the detection of erythromycin
FRET-based sensor system for the detection of erythromycin that consists of erythromycin C-12 hydroxylase (BBa_K4447001),an enzyme that catalyzes the oxidation of erythromycin, flanked by two fluorescent proteins: ECFP (BBa_K1159302)as an energy donor and Venus (BBa_K1907000)as an energy acceptor.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1913
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2562
Usage and Biology
With the rise of synthetic biology, biosensors have gained popularity over the years. Biosensors are devices that detect the presence of certain compounds by using living organisms or biological molecules.
In this composite part, we propose an enzymatic system based on how the Förster resonance energy transfer (FRET) operates: one enzyme capable of recognizing and degrading erythromycin will be flanked by two fluorescent proteins. This part incorporates NcoI and XhoI restriction sites in 5' and 3' ends for protein overexpression in pBAD/Myc-His plasmids, a gly-gly-ser spacer, and a polyhistidine tag before stop codon at the end of Venus for protein purification. Any linker does not separate each protein; for instance, stop codons for ECFP and erythromycin C-12 hydroxylase were removed. Figure 1 displays the three-dimensional structure of this protein system.
Selecting Fluorescent Proteins
Fluorescent proteins are most commonly used as donor and acceptor fluorophores in FRET biosensors, especially since these proteins are genetically encodable and live-cell compatible. For this section, we relied on the articles from Bajar et al. (2016) and Agrawal et al. (2021), where different fluorescent proteins are compared according to the requirements of a particular system. The particularity of fluorescent proteins depends on three main advantages: fluorescent proteins-based biosensors are easily constructed via genetic engineering, they confer high cellular specificity by using specific promoters, and these systems are stable in cells for a long time due to high intracellular stability.
From the pairs suggested by Bajar et al. (2016), enhanced cyan fluorescent protein (ECFP) and mVENUS (YFP) are widely recommended because of a higher quantum yield and better folding at 37 °C. This fact is also confirmed by Agrawal et al. (2021), who successfully developed a functional FRET-based sensor to monitor silver ions using this pair of fluorescent proteins. Agrawal et al. (2021) mention that the emission spectrum was recorded after excitation of the sensor protein at 420 nm, and recording the emission in the range of 450 to 600 nm, reaching a peak in 530 nm.
Sequences from both fluorescent proteins were obtained from the BioBricks catalog provided by iGEM. Finally, we selected these fluorescent proteins:
•BBa_K1159302: Enhanced Cyan Fluorescent Protein (ECFP). This Biobrick is an improved version of BBa_E0022, allowing protein fusion that was not initially possible by assembly criteria.
•BBa_K1907000: Venus. This part is a variant of yellow fluorescent protein, making it more stable and improving efficiency maturation.
Assembling Biobricks
Letsgp
References
[1]. Agrawal, N., Soleja, N., Bano, R., Nazir, R., Siddiqi, T. O., & Mohsin, M. (2021). FRET-Based Genetically Encoded Sensor to Monitor Silver Ions. ACS omega, 6(22), 14164–14173. https://doi.org/10.1021/acsomega.1c00741
[2]. Bajar, B., Lam, A., Badiee, R. et al. (2016). Fluorescent indicators for simultaneous reporting of all four cell cycle phases. Nat Methods 13, 993–996. https://doi.org/10.1038/nmeth.4045