Part:BBa_K4260111
RecA mini intein mediated ESR1 protein with OmpA signal peptide, linker and AmilCP.
This part encodes an intein mediated biosensor, consisting of a divided AmilCP chromoprotein gene, a mutated intein RecA N-terminal and C-terminal domains and a ESR1 Ligand Binding Domain. The RecA intein comes from Mycobacterium tuberculosis (Mtu) which has been mutated with the purpose of making it more stable.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design
RecA intein ESR1 biosensor consists of two inteins capable joining two protein fragments and separating from them. The ESR1 biosensor gene coding for the hERalpha protein was introduced with its respective linker, with the purpose of separating the N-terminal and C-terminal RecA inteins in the presence of EDCs; when the ESR1 biosensor binds to an EDC, the inteins may carry protein spicing, ligate the two N and C exteins and separate the ESR1 biosensor, ESR1 linker and the two N and C extein complex. The two endogenous fragments encode the chromoprotein AmilCP [BBa_K592009].
RecA intein was build from the first 111 and last 58 amino acids from the wild type RecA full-lenght intein [1]. Moreover a mutation was carried (Val67Leu) to make the RecA intein a stable protein [2]. The functionality of the Barcelona 2020 team's inteins from the biobrick [BBa_K348400] was verified to check the functibility of the HERα protein to be used in the biosensor as a biological receptor for EDCs.
The whole coding sequence consists of the OmpA solubility tag for better interaction between the biosensor and EDCs, the first 100 amino acids from the AmilCP blue chromoprotein [BBa_K592009], the N-terminal RecA mini intein consisting of the first 111 amino acids from Mycobacterium tuberculosis (Mtu) RecA intein with optimized codons for E. coli with a mutation (V67L) for stabilizing the structure that had been perturbed without a central endonuclease domain [6], the ESR1 linker to avoid interactions between proteins, the ESR1 biosensor, the C-terminal RecA mini intein consisting of the last 58 amino acids of Mtu RecA intein, and the rest of the AmilCP blue chromoprotein.
Usage and Biology
Biology
The RecA mini intein ESR1 biosensor was synthesized in two fragments: the first 533 bases as the first fragment and the last 2764 bases as the second fragment by IDT. An Overlapping PCR was carried, where two homologous sequences get overlapped and amplified with the objective of getting a single DNA strand [7]. Immediately the overlapping PCR was done, a normal amplification PCR was carried. The final PCR product was run into an agarose gel through electrophoresis and purified. The vector used to add the RecA intein biosensor was pJET 1.2/blunt and cloned into ''E.coli'' DH5-alpha strain [8]. After cloning, the plasmid was extracted and cloned into BL21 and HMS174 E. coli strains for protein expression.
Usage
Two protein expression plans were made with the purpose of exploring different types of protein expression, the first one was a Cell Free System, which does not require living cells to perform this expression, instead the cell machinery is used to express the wanted protein; the second one is a living cell system with solid and liquid media with different concentrations of EDCs. Plan 1: Cell Free System
Firstly, the BL21 strain was inoculated and incubated overnight in 100 mL of LB+Amp media, when the cells were at an optical density of 0.6, they were induced with IPTG and left overnight; then the cells were centrifuged, and the biomass was resuspended with R1 buffer, lysed with the help of the polytron homogenizer, and the supernatant was recovered containing protein remains and ribosomes. The resulting supernatant can be used to carry protein expression without the need for any transformed living cells since all the machinery needed to express protein such as ribosomes and protein remains are all present in the supernatant. After the cell “extract†was obtained, the RecA mini intein biosensor amplified sequence through PCR was added to the extract for its proper translation and expression, along with EDCs: three solutions were made, one with cell extract, PCR amplified sequence and EDCs, the second without the PCR amplified sequence, and the third without EDCs. The expected result is a change in coloration observed in the first solution, in contrast with the other two, that may serve as negative controls.
Plan 2: IntAssay After researching different ways to measure the functionality of a sensing protein HERα, it was found that the use of intein protein splicing and protein fusion of the former with natural binding-domains can certainly be used to sense synthetic molecules [2] and leave visible evidence of the functionality of the receptor protein, in our case HERα, and in further research it was found that a iGEM Team UPF Barcelona had done a similar measurement with thyroid hormones as ligand. Then the part BBa_K4260111 was designed, always contemplating the restriction sites established by iGEM. For the improvement of our gene expression and to have an efficient purification of the protein, we established an IPTG induction system and a signal peptide that led the translation into the periplasmic space along with RBS, a T7 promoter and terminator, synthesized by sponsor IDT. By expressing the part BBa_K4260111 inside pJET 1.2 obtained by Thermofisher Ⓡ, even though said vector is a cloning one, the sequence already had parts that allowed protein translation, and the advantage of the pJET 1.2/blunt killer switch was taken in account. The DNA sequence was synthesized into separated parts and united by a PCR overlapping and amplified after by PCR using iGEM primers. It was transformed within DH5α strain, the plasmid was extracted and characterized by restriction enzymes, then transformed again in HMS174 strain of E.coli, the reporter gen is meant to be expressed and free amilCP blue chromoprotein to be completed due to the splicing occurring between inteins when an EDC is caught by HERα. The quantity of blue chromoprotein is expected to vary increasingly as the concentration of BPA grows. This variation will be measured by an spectrophotometer, with LB medium as blank and the sample analysis of triplicated E.coli culture exposed to BPA concentrations after induction with IPTG. There was four different BPA concentrations managed in this assay, 1.69 mg/L, 1 mg/L, 5.5 mg/L, 11 mg/L , and a stock solution 100mgL , the range of the concentrations included the ones obtain in analysis of wastewater and the concentration at which the mitochondria stops being functional [] the assay divided in three ways to expose the cell to the solution BPA/ethanol, the first being plating 100 L of the solutions in petri dishes, doing triplicates and three negative controls containing ethanol, IPTG and bacteria alone. The second method was the solutions being added to 10 mL liquid LB medium with HMS174-BBa_K4260111 induced by IPTG and to observe the results. It was proposed to measure the variations of the intensity of the blue chromoprotein by absorbances at 588 nm and then treat them by ANOVA analysis to determine if the BPA concentration is relevant to the blue chromoprotein concentration in the medium. Unfortunately, the results were inconclusive due to the lack of protein expression, and the possibility of the inability of the construction to be spliced in the presence of EDCs was considered. In spite of this, different paths were designed to express it better, the sequence was ligated with pET17b expression vector by digesting said vector with ECORI-HF from New England BioLabs cutting in its MCS and then with a blunting enzyme eliminating the sticky ends to avoid scars, provided along with pJET 1.2 kit from Thermofisher, was utilized and the PCR results were ligated with the vector and afterwards transformed in HMS174 strain known for its ability to produce proteins from pET vectors. The assays were performed again but improving the biodisponibility of the cells to the BPA solutions and the grown cells inducted were centrifuged, resuspended in buffer, and exposed to BPA. A free cell system containing the machinery of BL21 strain, the PCR sequence and EDCs was done. All the assays failed to produce the desired effect of the intein splicing and liberation of blue chromoprotein.
References
[1] Wood, D. W., Wu, W., Belfort, G., Derbyshire, V., & Belfort, M. (1999). A genetic system yields self-cleaving inteins for bioseparations. Nature biotechnology, 17(9), 889-892.
[2] Gierach, I., Li, J., Wu, W. Y., Grover, G. J., & Wood, D. W. (2012). Bacterial biosensors for screening isoform-selective ligands for human thyroid receptors α-1 and β-1. FEBS open bio, 2, 247-253.
[3] Van Roey, P., Pereira, B., Li, Z., Hiraga, K., Belfort, M., & Derbyshire, V. (2007). Crystallographic and mutational studies of Mycobacterium tuberculosis recA mini-inteins suggest a pivotal role for a highly conserved aspartate residue. Journal of molecular biology, 367(1), 162-173.
[4] TecCEM 2021 https://2021.igem.org/Team:TecCEM
[5] Joshua S. Klein, Siduo Jiang, Rachel P. Galimidi, Jennifer R. Keeffe, Pamela J. Bjorkman. (2014) Design and characterization of structured protein linkers with differing flexibilities. Protein Engineering, Design and Selection, Volume 27, Issue 10, Pages 325–330. https://doi.org/10.1093/protein/gzu043
[6] Chen, X., Zaro, J. L., & Shen, W.-C. (2013). Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews, 65(10), 1357–1369. doi:10.1016/j.addr.2012.09.039
[7] Anna Bahle. (2019). Overlap extension PCR. CEPLAS, Heinrich Heine University. Institute for Synthetic Microbiology. Protocols.io. https://dx.doi.org/10.17504/protocols.io.psndnde
[8] pJET 1.2/blunt vector: https://www.snapgene.com/resources/plasmid-files/?set=basic_cloning_vectors&plasmid=pJET1.2
Costa, S., Almeida, A., Castro, A., & Domingues, L. (2014). Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system. Frontiers in microbiology, 5, 63.
Shah, N. H., & Muir, T. W. (2014). Inteins: nature's gift to protein chemists. Chemical science, 5(2), 446-461.
Shingledecker, K., Jiang, S. Q., & Paulus, H. (1998). Molecular dissection of the Mycobacterium tuberculosis RecA intein: design of a minimal intein and of a trans-splicing system involving two intein fragments. Gene, 207(2), 187-195.
Davis, E. O., Sedgwick, S. G., & Colston, M. J. (1991). Novel structure of the recA locus of Mycobacterium tuberculosis implies processing of the gene product. Journal of bacteriology, 173(18), 5653-5662.
| None |