Part:BBa_K4260111
RecA mini intein mediated ESR1 protein with OmpA signal peptide, linker and AmilCP.
This part encodes an intein mediated protein, 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.
The usage of an intein mediated protein allows for protein post traductional modification in presence of a ligand, in this case an EDC, through a process known as protein splicing, consisting of a multi-step biochemical reaction comprised as a cleavage and the formation of a peptide bond. A change in coloration depending on the EDC concentration offers a visual indicator due to the union of the two N and C extein, being a AmilCP chromoprotein [BBa_K592009]. The expected result being a blue coloration in the final media or after the protein centrifugation.
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 protein consists of two inteins capable joining two protein fragments and separating from them. The ESR1 protein 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 protein binds to an EDC, the inteins may carry protein spicing, ligate the two N and C exteins and separate the ESR1 protein, 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 as a biological receptor for EDCs.
The whole coding sequence consists of the OmpA solubility tag for better interaction between the protein 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 protein, 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 protein 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 mini intein protein 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 protein 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
The quantity of blue chromoprotein is expected to vary increasingly as the concentration of BPA grows. This variation will be measured by a spectrophotometer, with LB medium as blank and the sample analysis of triplicated E. coli culture exposed to BPA concentrations after induction with IPTG. There were four different BPA concentrations managed in this assay, 1.69 mg/L, 1 mg/L, 5.5 mg/L, 11 mg/L, the range of the concentrations included the ones obtain in analysis of wastewater [9] and the concentration at which the mitochondrial complex I decreases [10]. 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 media.
Results
Overlapping PCR
The overlapping PCR was carried to ligate two RecA mini intein ESR1 protein sequences. With a successful ligation and amplification of the resulting ligated DNA sequence. The two initial sequences had a homologous region required to carry the overlapping PCR protocol; the first sequence (RecA seq. 1) had a length of 533 bp and the second one (RecA seq 2.) had 2764 bp, resulting on an expected 3297 bp ligated DNA sequence.
pJET1.2/blunt vector ligation
The resultant PCR amplification was purified and later ligated into pJET1.2/blunt since this vector facilitates ligation due to its blunt ends and offers ampicillin resistance. The resulting plasmid was transformed into DH5-alpha and plated in petri dish with LB+Ampicilin media.
After transformation, a colony was inoculated with liquid LB+Ampicilin media and a plasmid extraction was performed using from GenElute™ Plasmid Miniprep Kit. The extracted plasmid was run into an agarose electrophoresis to verify its integrity and desired size.
Protein expression plans result table
Plan | Description | Expected results | Experimental results | Proposed implementation |
Cell Free System | The Cell Free System was carried with a lysed cell extract, where the cell machinery (ribosome) is used to express our amplified PCR product RecA mini intein-ESR1 protein | Blue coloration after several hours due to transcription and traduction in the sample. Included lysed cell extract, PCR mix, BPA and R1 buffer. | Did not show any kind of blue coloration past 24 hours. | Try with different lysis protocol and more concentrated PCR sample. |
pJET1.2/blunt transformation into HMS174 | This pJET1.2/blunt transformation was done in the E.coli HMS174 strain, it was inoculated, induced, and lysed. After lysis, BPA was added to each sample and monitored every hour. | Blue coloration after several hours, or a blue pellet after centrifugation. | Every sample showed no blue coloration after more than 24 hours. | Change strain |
IntAssay | The RecA mini intein ESR1 was ligated into the pET-17b plasmid with the help of a blunting enzyme and transformed into E.coli HMS174. 21 tubes were inoculated and induced to carry the IntAssay, adding BPA in several concentrations. | Blue coloured media after BPA addition. | No blue coloration was observed after more than 24 hours of the addition of BPA. | Change strain, |
The lack of coloration is hypothesized to have occurred due to an interaction affecting its ability to perform protein splicing; this can be observed in a molecule docking between complexes. At the same time, the usage of the E.coli HMS174 strain may have been faulty or deficient in its protein expression.
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
[9] Fukazawa, H., Hoshino, K., Shiozawa, T., Matsushita, H., & Terao, Y. (2001). Identification and quantification of chlorinated bisphenol A in wastewater from wastepaper recycling plants. Chemosphere, 44(5), 973-979.
[10] Ooe H., Taira T., Iguchi-Ariga S. M. and Ariga H. (2005) Induction of reactive oxygen species by bisphenol A and abrogation of bisphenol A-induced cell injury by DJ-1. Toxicol Sci 88, 114-26.
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