Part:BBa_K4260006
IIsoeugenol Monooxygenase, autoinducible coding sequence: Trp terminator, IemR, promoter, pelB, Iso and rrnB T1 terminator.
Type:Composite part
Designed by:Claudia Angélica GarcÃa Alonso
Group:iGEM_TecCEM
IBBa_K4260010 is a composite part that aims to work as a selection marker for the replacement of antibiotic-based selection markers. Isoeugenol monooxygenase (coded by gene Iso) is an enzyme present in Pseudomonas bacteria that converts isoeugenol to vanillin directly. Its transcription is regulated by the expression of its transcriptional activator (IemR), as well as the presence of isoeugenol. Isoeugenol is an aromatic compound found in clover and cinnamon essential oils, that has proved to have an inhibitory effect on different microorganisms; however, this genetic construct was designed for its expression in Escherichia coli. Its antimicrobial activity resides in altering the integrity of the inner membrane of the bacteria, leading to fluid spillage and cell death. Therefore, by directing the enzyme to the periplasm of the bacteria, only transformed cells could survive when exposed to isoeugenol.
Design
This is a 2807 bp long sequence, that includes a bidirectional promoter that regulates the transcription of Iso (coding gene of IsoMo) and IemR (a transcriptional activator reported by Ryu, Seo, Park, Ahn, Chong, Sadowsky & Hur in 2012, GenBank access code: FJ851547.1) [1], in the 5’ to 3’ and 3’ to 5’ direction, respectively. The evaluation of potential inducers of the promoter from Pseudomonas nitroreducens Jin1 was evaluated by the same authors, who came to the conclusion that isoeugenol was the best of all candidates. Lastly, two different terminators were added, one for each coding gene: Trp (BBa_K4260008) [2] and rrnB T1. No codon optimization was performed on either the promoter nor the terminator sequences. Also, Leu359 was changed from CTG to CTC in order to eliminate a PstI restriction site and make sure that the prefix restriction sites (EcoRI and XbaI) and suffix restriction sites (SpeI and PstI) appeared only once in the entire biobrick.
Inducible promoter + transcriptional activator
The transcription of the isoeugenol monooxygenase (iso) is regulated mainly by the protein encoded by the gen iemR (from the strain P. nitroreducens JinI), and by the presence of isoeugenol in the medium. iemR is a positive transcriptional regulator gene which is upstream of iem and, according to Ryu, J. Y. et al. (2012), its overproduction increases the transcription of the isoeugenol monooxygenase.
This regulator gene works along with the putative iem promoter, and according to Ryu, J. Y. et al. (2010), the production of the monooxygenase was increased by the presence of isoeugenol in the medium, suggesting that this gene regulates the expression of iem based on the presence of the isoeugenol.
Isoeugenol works as the substrate of the iemR encoded protein and regulator, and a helix-turn-helix motif was found in the molecule of this inductor, which corresponds to regulators from the family of AraC/XyIS. According to Tropel, D. et al. (2004), this kind of regulators act as activators of gene transcription when some chemical compound is present, in this case the isoeugenol as antibiotic.[3] Also, the expression of this type of regulator genes can happen thanks to a dependent promoter, which in this case is the putative iem promoter. The section XyIS is capable to bind to two repeated sequences in the promoter’s sequence, by forming one dimer or two monomers. This demonstrates the interaction between the gene promotor and the inductor protein thanks to the presence of isoeugenol, which would allow the correct expression of our gene of interest.
Characterization
The active site of the enzyme, according to Ryu, Seo, Park, Ahn, Chong, Sadowsky & Hur (2013) [1], is integrated by residues: H167, H218, H282, H471, E135, E349 & E413. As shown in Fig. 2 it is interiorized.
Another important feature of the IsoMo, is the energy heatmap for IsoMo shows that it has good values of ionic strength in a pH range of 4-8, indicating good tolerance to pH changes (Fig 3.) at most of its amino acids, which can be convenient for further usage of this part, given that the culture of certain microorganisms leads to variations on the pH of the culture; however, it is important to point out that the better value of energy occurs at a pH of 8, which matches information reported in BRENDA [3].
Figure 3 shows that the IsoMo from a pH of 4, to lower values, the ionic strength becomes dependent, that is, if the pH decreases, the ionic strength increases, causing a possible loss of stability due to loss of protein solubility due to a salting-out effect. On the other hand, in figure 4 it can be seen that the isoelectric point of the protein is at a pH of 6 in a concentration range of ionic strength from 0.005 to 0.15 M, which corresponds to the theoretical isoelectric point of 5.71 obtained from the expasy software of Swiss Bioinformatics Resource Portal.
Figure. 4 Charge heatmap of Isoeugenol Monooxygenase, per aminoacid.
Figure 5 is a representative image of the results that can be observed in figure 3, of which areas of the enzyme have positive and which one have negative charges based on the pH of the medium where the enzyme is in contact with.
Figure 6 a 3D model of the representation of the hydrophobicity of IsoMo, in this image it can be observed un green color the parts hydrophobic and the purple are the hydrophilic sites.
Usage and biology
In particular this sequence was inserted in 2 E coli strains, DH5α and BL21, for the ligation pJET was used, as we were aiming to characterize our composite part, all transformed cells were grown in LB media with Ampicillin to ensure that all the bacteria will inherit the vector. After making the transformation we get 7 inserts, 5 in DH5α and 1 BL21,, to which we wanted to carry out a series of experiments in order to analyze which of them was the best and in which strain they could be expressed and cloned in a more optimal way. For analyzing that the transformation of these strains in those strains was successful. First of all a plasmid extraction was performed and with the help of an electrophoresis gel we proved that the plasmid was there. In figure 7 we can observe the presence of the 4 extracted plasmids, no contamination was observed.
As the electrophoresis gel does not show a clear idea if the plasmid was there, with that samples, we carried out a series of restriction enzyme digestions so that we can confirm the presence of the plasmid, for this we review the restriction map of the plasmid, to help us identify the cutting sites of the enzymes and the bp of that cut, so for this particular plasmid, XbaI and SpeI were chosen for performing the digestions, this result is shown in figure 8.
After verifying the presence of Pp_22 in the transformed cells and having characterized the plasmid (pJET) with the insert, we selected 2 of the 7 samples we had, the ones that we identify of being more present in the results shown before were: E. coli DH5αtransformed with Pp_22 (samples, as identify in the gels, 4 and 5). Once having all this established, an analysis of expression was the next step; for this aim, IsoMo expression was evaluated by culturing BL21 cells containing the Nc sequence in LB medium and a synthetic medium, at different concentrations of isoeugenol. For this, a total protein extraction samples were used in SDS-PAGE. As a control in this experiment, not transformed BL21 E. coli strains were also cultured under the same conditions.
After performing an SDS-Page for analyzing the expression of IsoMo, there was no expression shown, so it was determined that the plasmid (pJET) used was not, very useful for the expression of the enzyme, since it is a cloning vector.
helping the expression of the enzyme, because this is a plasmid that helps cloning the sequence but not expressing the protein. Finally, the analysis of its interaction with its substrate is shown below, where it can be ween that a hydrogen bond is formes with Methionine 350.
Application
This part is intended to be an alternative selection marker, for replacing the usage of antibiotic in the techniques perform in the laboratories of synthetic biology.
Biosafety
Although this coding sequence comes from a Pseudomona bacteria, it is not associated with the pathogenicity of the microorganism itself.
References
[1] Ryu, J. Y., Seo, J., Ahn, J. H., Sadowsky, M. J., & Hur, H. G. (2012). Transcriptional control of the isoeugenol monooxygenase of Pseudomonas nitroreducens Jin1 in Escherichia coli. Bioscience, biotechnology, and biochemistry, 76(10), 1891-1896.
[2] https://parts.igem.org/Part:BBa_K4260008
[3] Tropel, D. et al. (2004). Bacterial Transcriptional Regulators for Degradation Pathways of Aromatic Compounds. MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, p. 474–500. DOI: 10.1128/MMBR.68.3.474–500.2004.
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