Parts
To construct our parts, we used different approaches. Some were synthesised by IDT, others were obtained by PCR amplification from plasmids such as pBAD, pLO_SNAP and pET28.
Basic and composite parts
| Part code | Name | Type | Description | Length |
|---|---|---|---|---|
| BBa_K4345000 | NarX fused to mNeonGreen with a rigid linker | Composite part | Three proteins constitute a two-component nitrate sensing system in Escherichia coli: NarX, NarL and NarQ. Together they are responsible for the expression of anaerobic respiratory genes. NarX and NarQ are histidine kinases that independently detect the presence of nitrate an transmit the signal to NarL. After autophosphorylation and thus dimerization of NarX or NarQ, NarL is phosphorylated. This enables the activated NarL to bind DNA and induce expression of specific genes. Both NarX and narQ contain two conserved histidine residues that correspond to the autophosphorylation sites of other, homologous, sensor-transmitter proteins (Cavicchioli et al., 1995). Cheung & Hendrickson (2009) elucidated the structure of the histidine kinase in the apo- and holo-state to be a four-helix bundle. To follow the expression of NarX, it is fused to mNeonGreen with a rigid linker. This part is composed of BBa_K4345007 (NarX), BBa_K4345002 (rigid linker) and BBa_K4345009 (mNeonGreen). | 2594 bp |
| BBa_K4345001 | RNA Thermometer hsp17 | Basic part | Hsp17 is in nature implemented in the mRNA structure of a cyanobacterial (Synechocystis sp.) heatshock gene which is important for stress tolerance. The thermometer is a structural element that is located within the 5’ UTR of protein-coding mRNA. It operates as a reversible molecular zipper that controls the availability of the RBS in its structure. When the temperature increases, the thermometer unzips and the RBS can be detected by the ribosome thus allowing translation of downstream mRNA. When the temperature is below a certain range, the RBS gets trapped. The 5’ UTR end consists of a single hairpin with an internal asymmetric loop, as seen in the figure below. The secondary structure was predicted by RNAFold. The trapped RBS sequence is highlighted in blue. The start codon is highlighted in green and is located 45 nucleotides downstream from the transcriptional start site which means that the thermometer is very short compared to others that are described. | 50 bp |
| BBa_K4345002 | Rigid linker | Basic part | This linker allows for the independent folding of both proteins it fuses together. | 75 bp |
| BBa_K4345003 | mPapaya | Basic part | mPapaya is a constitutively fluorescent protein with an exctitation wavelength of 530 nm and an emission wavelength of 541 nm. | 711 bp |
| BBa_K4345004 | NarX mutant fused to mPapaya with a rigid linker | Composite part | For more information on NarX, go to this part page: BBa_K4345000. The mutant was designed based on a study of Cavicchioli et al., (1995). The histidine on place 399 was replaced by glutamic acid. This allows the NarX mutants to dimerize but blocks it from phosphorylating NarL (the second messenger). This composite part consists of BBa_K4345008 (NarX H399-E), BBa_K4345002 (Rigid linker) and BBa_K4345003 (mPapaya). | 2597 bp |
| BBa_K4345005 | T7 terminator | Basic part | This bacteriophage T7 terminator allows for efficient termination by forming a stem-loop structure followed by a run of six uridylate residues. | 84 bp |
| BBa_K4345006 | mKate2 | Basic part | mKate2 is a red constitutively fluorescent protein with an exctitation wavelength of 588 nm and an emission wavelength of 633 nm. | 699 bp |
| BBa_K4345007 | NarX | Basic part | NarX is a part of NarX-NarL two component nitrate sensing system in Escherichia coli. They are responsible for the expression of anaerobic respiratory genes. NarX is a histidine kinases that independently detect the presence of nitrate an transmit the signal to NarL. After autophosphorylation and thus dimerization of NarX or NarQ, NarL is phosphorylated. This enables the activated NarL to bind DNA and induce expression of specific genes. Both NarX and narQ contain two conserved histidine residues that correspond to the autophosphorylation sites of other, homologous, sensor-transmitter proteins (Cavicchioli et al., 1995). Cheung & Hendrickson (2009) elucidated the structure of the histidine kinase in the apo- and holo-state to be a four-helix bundle. | 1794 bp |
| BBa_K4345008 | NarX H399-E | Basic part | The mutant was designed based on a study of Cavicchioli et al., (1995). The histidine on place 399 was replaced by glutamic acid. This allows the NarX mutants to dimerize but blocks it from phosphorylating NarL (the second messenger). | 1797 bp |
| BBa_K4345009 | mNeonGreen | Basic part | mNeonGreen is a constitutively yellow/green fluorescent protein with an excitation wavelength of 506 nm and an emission wavelength of 517 nm. | 711 bp |
| BBa_K4345010 | RNA thermometer prfA | Basic part | RNA thermometer used for post-transcriptional regulation based on a temperature-dependent system, based on a RNA thermometer from Listeria monocytogenes. It is maximally expressed at 37ºC and almost not detectable at 30ºC. | 130 bp |
| BBa_K4345011 | TT3-rrnD1-T1 | Basic part | Transcription terminator T1 from the E. coli rrnB gene. This terminator is a part of Naringenin-Responsive transcriptional biosensor develop by De Paepe et al. (2018). | 90 bp |
| BBa_K4345012 | fdeR | Basic part | Coding sequence of fdeR from PfdeAR-FdeR biosensor circuit's detector module. FdeR is a part of Naringenin-Responsive transcriptional biosensor develop by De Paepe et al. (2018). Native naringenin-responsive regulatory circuit, LysR-type PfdeAR-FdeR pair from Herbaspirillum seropedicae was redesigned to study the naringenin response in E. coli. | 930 bp |
| BBa_K4345013 | PfdeAR | Basic part | PfdeAR is a native bidirectional promotor of LysR-type PfdeAR-FdeR pair from Herbaspirillum seropedicae. PfdeAR is a part of PfdeAR-FdeR biosensor circuit from the naringenin-responsive transcriptional biosensor develop by De Paepe et al. (2018). | 141 bp |
| BBa_K4345014 | rrnB T1 terminator | Basic part | Transcription terminator T1 from the E. coli rrnB gene. This terminator is a part of Naringenin-Responsive transcriptional biosensor develop by De Paepe et al. (2018). | 72 bp |
| BBa_K4345015 | pSynSens2.5000 RBS | Basic part | This RBS was developed using a library and optimised to work robustly in pSynSense2.5000 designed by De Paepe et al. (2018). | 35 bp |
| BBa_K4345016 | ccdB with STOP codon control | Basic part | CcdB protein is toxin of type II toxin-antitoxin system CcdB/CcdA. CcdB is a toxin that kills E. coli by targeting the GyrA subunit of DNA gyrase, an essential type II topoisomerase (Bahassi et al., 1999). In this part, we added three stop codons to prevent the translation and be able to clone CcdB into our host. | 342 bp |
| BBa_K4345017 | pSynSense2.5000 PfdeAR-FdeR biosensor circuit | Composite part | Biosensor circuit in pSynSense2.5000 developed by De Paepe et al. (2018) is composed of TT3-rrnD1-T1, fdeR, rrnB T1 terminator, PFdeAR, and pSynSens2.5000 RBS. The modified LysR-type PfdeAR-FdeR pair from Herbaspirillum seropedicae allows this circuit to respond to naringenin. There are two modules in the circuit: detector and effector. In the detector module, FdeR will produce the transcription factor of the effector module. This transcription factor will bind to the binding site upon the presence of naringenin, thus making this circuit naringenin responsive. | 1713 bp |
| BBa_K4345018 | Tet/lac promoter - ccdB fused to sfGFP | Composite part | The toxin ccdB (including the stop codon control, see BBa_K4345018) is under the control of a promoter that is activated by both anhydrotetracyclin and lacI. ccdB is fused to sfGFP with a rigid linker. | 1315 bp |
| BBa_K4345019 | RNA Thermometer rpoH | Basic part | " In nature, rpoH is implemented in the mRNA structure of a Escherichia Coli heatshock gene which is important for stress tolerance. The thermometer is a structural element that is located within the 5’ UTR of protein-coding mRNA. It operates as a reversible molecular zipper that controls the availability of the RBS in its structure. When the temperature increases, the thermometer unzips and the RBS can be detected by the ribosome thus allowing translation of downstream mRNA. When the temperature is below a certain range, the RBS gets trapped. | 248 bp |
| BBa_K4345020 | 5' UTR of hsp17 with ccdA fused to sfGFP | Composite part | Design of a thermoregulated translation of antitoxin ccdA. Fluorescent protein sfGFP was linked to the antitoxin with a rigid linker for visualisation. | 947 bp |
| BBa_K4345021 | LacO | Basic part | The Lac operator is a crucial part for the transcription of downstream genes. When a repressor is present (like LacI), the RNA polymerase is not able to do transcription of the DNA. This repression can be lifted by IPTG and thus induce the circuit. | 17 bp |
| BBa_K4345022 | Tet promoter with lac operator | Composite part | This promoter was designed to be both controlled by anhydrotetracyclin and lacI. Both are positive regulators. | 74 bp |
| BBa_K4345023 | 5' UTR of rpoH with ccdA fused to sfGFP | Composite part | Design of a thermoregulated translation of antitoxin ccdA. Fluorescent protein sfGFP was linked to the antitoxin with a rigid linker for visualisation. | 1359 bp |
| BBa_K4345024 | 5' UTR of prfA with ccdA fused to sfGFP | Composite part | Design of a thermoregulated translation of antitoxin ccdA. Fluorescent protein sfGFP was linked to the antitoxin with a rigid linker for visualisation. | 1277 bp |
Part collection
Biosensors have been a simple but accurate tool for analytical purposes for a while. They respond fast for measurements of chemical or biological markers in different applications. It is then relevant to make sure that the indispensable parts of the biosensors are designed adequately. Our Part Collection focuses on some basic and composite parts that are essential for the design of a biosensor. As biorecognition elements we designed BBa_K4345004 and BBa_K4345000 (three proteins that constitute a two-component nitrate sensing system in Escherichia coli: NarX, NarL and NarQ). The transducer of this system is based on BBa_K4345009, being it the transducer in order to be able to later on detect the fluorescence emission. Another part essential for biosensors are the kill switches. We developed them based on RNA thermometers of the proteins hsp17, rpoH and prfA.
References
- Bahassi, E. M., O’Dea, M. H., Allali, N., Messens, J., Gellert, M., & Couturier, M. (1999, April). Interactions of CcdB with DNA Gyrase. Journal of Biological Chemistry, 274(16), 10936–10944. https://doi.org/10.1074/jbc.274.16.10936
- Cheung, J., & Hendrickson, W. A. (2009, February). Structural Analysis of Ligand Stimulation of the Histidine Kinase NarX. Structure, 17(2), 190–201. https://doi.org/10.1016/j.str.2008.12.013
- Johansson, J., Mandin, P., Renzoni, A., Chiaruttini, C., Springer, M., & Cossart, P. (2002, September). An RNA Thermosensor Controls Expression of Virulence Genes in Listeria monocytogenes. Cell, 110(5), 551–561. https://doi.org/10.1016/s0092-8674(02)00905-4
- Kortmann, J., Sczodrok, S., Rinnenthal, J., Schwalbe, H., & Narberhaus, F. (2010, December 3). Translation on demand by a simple RNA-based thermosensor. Nucleic Acids Research, 39(7), 2855–2868. https://doi.org/10.1093/nar/gkq1252
- Lambert, T. (n.d.). mNeonGreen at. FPbase. Retrieved September 27, 2022, from https://www.fpbase.org/protein/mneongreen/
- mKate2. (2022). FPBase. Retrieved July 7, 2022, from https://www.fpbase.org/protein/mkate2/
- mNeonGreen. (2022). FPbase. Retrieved July 7, 2022, from https://www.fpbase.org/protein/mneongreen/
- Morita, M. T., Tanaka, Y., Kodama, T. S., Kyogoku, Y., Yanagi, H., & Yura, T. (1999, March 15). Translational induction of heat shock transcription factor sigma 32: evidence for a built-in RNA thermosensor. Genes &Amp; Development, 13(6), 655–665. https://doi.org/10.1101/gad.13.6.655
- mPapaya. (2022). FPBase. Retrieved July 7, 2022, from https://www.fpbase.org/protein/mpapaya/
- mPapaya1. (2022). SnapGene by Dotmatics. Retrieved July 7, 2022, from https://www.snapgene.com/resources/plasmid-files/?set=fluorescent_protein_genes_and_plasmids&plasmid=mPapaya1
- narX sensor histidine kinase NarX [ Escherichia coli str. K-12 substr. MG1655 ]. (2022, September 22). National Library of Medicine - National Center for Biotechnology Information. https://www.ncbi.nlm.nih.gov/gene/945788