Coding

Part:BBa_K4447004

Designed by: Rafael Garcia Gomez   Group: iGEM22_TecMonterrey_GDL   (2022-09-29)
Revision as of 21:44, 9 October 2022 by Rafa1470 (Talk | contribs) (→‎Usage and Biology)


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1913
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE 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. Among all of the biosensors available, Förster resonance energy transfer (FRET) biosensors are a powerful approach for dynamically tracking the presence of a particular substrate.

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.

Figure 1. Three-dimensional structure of the EryK-FRET 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.

Characterization

Restriction Enzyme Digestion and Ligation

We performed a restriction enzyme digestion with the synthesized part and the vector defined. Our digestion involved using NcoI and EcoRI restriction enzymes. For both vector and insert, DNA concentration was stated as 4000 nanograms. Table 1 displays the protocol followed for a 50 µL reaction.

Table 1. Restriction digest conditions.
Reactive Quantity
Nuclease-free water add to 50 µL
rCutSmart Buffer 5 µL
Template DNA (up to 4000 ng) X µL
NcoI restriction enzyme 1 µL
XhoI restriction enzyme 1 µL
Figure 3. Escherichia coli TOP10 colonies transformed with BBa_K4447004 cloned in pBAD/Myc-HisB. Bacteria were grown in LB medium with carbenicillin.

With the DNA fragments purified from an agarose gel, we performed ligation at a molar ratio of 1:5 for vector and insert, as shown in Figure 3. The total vector concentration was 100 nanograms, whereas the reaction volume was 20 µL. Next, Table 2 displays the protocol followed for the reaction.

Table 2. DNA ligation conditions.
Reactive Quantity
T4 DNA Ligase Buffer (10X) 2 µL
Vector DNA 100 ng
Insert DNA 773.5 ng
Nuclease-free water up to 20 µL
T4 DNA Ligase 1.5 µL

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
[edit]
Categories
Parameters
None