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]
Characterization
Usage and biology
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In particular this sequence was inserted in 2 E coli strains, DH5 and BL21, for the ligatión pJET was used, as we were aiming to characterize our IsoMo enzyme, all transformed cells were grown in LB media with Ampicillin to ensure that all the bacteria will inherit the vector. For analyzing that the transformation of Nc_22 in those strains was successful we performed a series of experiments; first of all a plasmid purificatión was performed and with the help of an electrophoresis gel we proved that the plasmid was there. |
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In particular this sequence was inserted in 2 E coli strains, DH5 and BL21, for the ligatión pJET was used, as we were aiming to characterize our IsoMo enzyme, all transformed cells were grown in LB media with Ampicillin to ensure that all the bacteria will inherit the vector. For analyzing that the transformation of Nc_22 in those strains was successful we performed a series of experiments; first of all a plasmid purificatión was performed and with the help of an electrophoresis gel we proved that the plasmid was there. |
This helped confirm the affinity of the enzyme for its substrate, as well as the interaction between the two molecules. After observing these results, it was determined that Iso coding sequence was a good option for the expression of isoeugenol monooxygenase.
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.
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