Composite
Pp_22

Part:BBa_K4260012

Designed by: Claudia Angélica García Alonso   Group: iGEM22_TecCEM   (2022-10-09)
Revision as of 06:48, 12 October 2022 by SofiaCN (Talk | contribs)


IIsoeugenol Monooxygenase, autoinducible coding sequence: Trp terminator, IemR, promoter, pelB, Iso and rrnB T1 terminator.


Type:Composite part

Designed by:Claudia Angélica García Alonso

Group:iGEM_TecCEM


IBBa_K4260006 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. Since the substrate of the enzyme is isoeugenol and the inducer of its promoter is also isoeugenol, it can be considered an auto-inducible transcription regulation system. 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.


Fig. 1 Designs scheme of the elements of BBa_K4260006.

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.


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.


Fig. 2 Active site of IsoMo. As it can be observed, it is mainly integrated by Histidine (H) residues and Glutamic acid (E).

Usage and biology

Figure 5:Electrophoresis gel plasmid extracción: 1) Quick-load 1 kb Extended, 2) Pp DH5α 1, 3) Pp DH5α 2, 4) Pp DH5α 3, 5) Pp DH5α 4, 6) Pp DH5α 5 and 7) Pp BL21.




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 5 we can observe the presence of the 4 extracted plasmids, no contamination was observed.

Figure 6:Electrophoresis gel digestions, with XbaI and SpeI: ) Quick-load 1 kb Extended, 2) Pp DH5α 1, 3) Pp DH5α 2, 4) Pp DH5α 3, 5) Pp DH5α 4, 6) Pp DH5α 5 and 7) Pp BL21.




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 6.


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.


File:SDS BB06.png.png
Figure 7:SDS-Page protein expression: 1)Marker, 2)DH5α, 3) Pp DH5α 4, 4) Pp DH5α 5.

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.

Application

For more information about the enzyme and its possible application as a selection marker, please check out BBa:K4260007.BBa:K4260006.

Biosafety

Although this coding sequence comes from a Pseudomona bacteria, it is not associated with the pathogenicity of the microorganism itself.


References

[1] Yamada, M., Okada, Y., Yoshida, T., & Nagasawa, T. (2008). Vanillin production using Escherichia coli cells over-expressing isoeugenol monooxygenase of Pseudomonas putida. Biotechnology letters, 30(4), 665-670.

[2] https://parts.igem.org/Part:BBa_K4260008

[3] https://parts.igem.org/Part:BBa_J32015

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