Part:BBa_K4260007
Isoeugenol monooxygenase, normal coding sequence: Promoter +RBS+pelB+Iso+rrnB T1 terminator
Type:Coding sequence
Designed by:Claudia Angélica GarcÃa Alonso
Group:iGEM_TecCEM
It is well known that antibiotics are necessary in synthetic biology experimental procedures. Therefore iGEM TecCEM created BBa_K4260007 a sequence designed for the replacement of tradicional selection markers, antibiotics; being our main focus the most commonly used microorganism Escherichia coli; in order to prevent more serious problems in the future due to antibiotic-resistant microorganisms. The sequence encodes the gene of Isoeugenol Monooxygenase (IsoMo), that allows bacteria to direct the enzyme to the periplasm with pelB signal sequence. With IsoMo we aim to confer bacteria the ability to resist isoeugenol, an inhibitory agent added to culture mediums. Isoeugenol acts on the inner membrane of bacteria, making it lose its integrity and causing the cell to spill out.
Design
In this composite part there can be found a modified gene, by codon optimization, from Pseudomonas putida IE27, reported by Yamada, Okada, Yoshida & Nagasawa in 2008 [1], Isoeugenol monooxygenase (IsoMo), that makes able to metabolize this compound, which allows a one-step conversion of it into vanillin, as well as pelB and an RBS, using a registered part from iGEM_TecCEM (BBa_K4260011) [2]. Within this part there is also a constitutive promoter for ensuring the transcription of genes despite any other conditions, for this aim we use (BBa_J23100) [3], as it has been reported this promoter has been characterized as one of the most efficient constitutive promoters. For the terminator another registered part was used rrnB (BBa_B0010). [4]
Constitutive promoter family (BBa_J23100)
As it is reported this family of promoters have been characterized as one of the most efficient promoters. As the main focus of this part is to be a selection marker, we needed to be activated the whole time, so for the focus of this kind of design we wanted to make a sequence that would make it easier for E.coli to able to transcribe and express constantly our enzyme of interest, isoeugenol monooxygenase. So with this type of constitutive promoter we can modify the expresión level of the part. [3]
Characterization
The active site of the enzyme, according to Ryu, Seo, Park, Ahn, Chong, Sadowsky & Hur (2013) [5], 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 [6].
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.
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 4 inserts, 2 in each of the E coli strains strains mentioned above, 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.
As the electrophoresis gel does not shows 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 these particular plasmid, XbaI and SpeI were chosen for performing the digestions, this result id shown in figure 6.
After verifying the presence of Nc_22 in the transformed cells, and having characterized the plasmid (pJET) with the insert, we selected 2 of the 4 samples we had, the ones that we identify of being more present in the results shown before were: E. coli BL21 transformed with Nc_22 (samples, as identify in the gels, 1 and 2). 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.
helping the expression of the enzyme, because this is a plasmid that helps cloning the sequence but not expressing the protein.Application
his 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] 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_B0030
[3] https://parts.igem.org/Part:BBa_J32015
[4] https://parts.igem.org/Part:BBa_B0010
[5] 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.
[1] BRENDA
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