Part:BBa_K5043011
amilGFP transcriptional unit
This part is a transcriptional unit, coding for amilGFP using Anderson promoter J23110, standard RBS B0034 and Terminator Luz7-T50. It was designed to verify protein production in Pseudomonas species.
Usage and Biology
Pseudomonas putida KT2440 [1] as well as Pseudomonas vancouverensis DSM8368 [2] show promising abilities as chassis for bioremediation of organic pollutants. [3, 4] In this context, we were interested in a low/medium constitutive expression system working in both bacteria. Hence, we decided to use Anderson promoter J23110, as it leads to medium expression in P. putida KT2440. [5] In addition, we chose RBS B0034 and Terminator Luz7-T50 both showing good efficiency in P. putida KT2440. [6, 7] As we could not find literature about these regulatory elements’ function in P. vancouverensis, amilGFP was used as reporter protein for qualitive assessment of protein production using this expression system. For this purpose, the shown composite part was cloned into pSEVA231-backbone. [8] Pseudomonas species were transformed using electroporatoration. Cell pellets of transformed liquid cultures exhibited no visible green colour under daylight, but green fluorescence under blue light.
As visible in the pictures, bacterial cultures bearing this composite part show green fluorescence, indicating GFP production. Thus, promoter J23110 and RBS B0034 both function in both P. putida KT2440 and P. vancouverensis DSM8368. The expression system is working.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 161
Illegal AgeI site found at 622 - 1000COMPATIBLE WITH RFC[1000]
References
[1] M. Bagdasarian et al., "Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas," Gene, vol. 16, 1-3, pp. 237–247, 1981, doi: 10.1016/0378-1119(81)90080-9.
[2] W. W. Mohn, A. E. Wilson, P. Bicho, and E. R. Moore, "Physiological and phylogenetic diversity of bacteria growing on resin acids," Systematic and applied microbiology, vol. 22, no. 1, pp. 68–78, 1999, doi: 10.1016/S0723-2020(99)80029-0.
[3] Z. Zuo et al., "Engineering Pseudomonas putida KT2440 for simultaneous degradation of organophosphates and pyrethroids and its application in bioremediation of soil," Biodegradation, vol. 26, no. 3, pp. 223–233, 2015, doi: 10.1007/s10532-015-9729-2.
[4] Y. Yang, R. F. Chen, and M. P. Shiaris, "Metabolism of naphthalene, fluorene, and phenanthrene: preliminary characterization of a cloned gene cluster from Pseudomonas putida NCIB 9816," Journal of bacteriology, vol. 176, no. 8, pp. 2158–2164, 1994, doi: 10.1128/jb.176.8.2158-2164.1994.
[5] A. N. Pearson et al., "The pGinger Family of Expression Plasmids," Microbiology spectrum, vol. 11, no. 3, e0037323, 2023, doi: 10.1128/spectrum.00373-23.
[6] E.-M. Lammens, L. Putzeys, M. Boon, and R. Lavigne, "Sourcing Phage-Encoded Terminators Using ONT-cappable-seq for SynBio Applications in Pseudomonas," ACS synthetic biology, vol. 12, no. 5, pp. 1415–1423, 2023, doi: 10.1021/acssynbio.3c00101.
[7] S. G. Damalas, C. Batianis, M. Martin-Pascual, V. de Lorenzo, and V. A. P. Martins Dos Santos, "SEVA 3.1: enabling interoperability of DNA assembly among the SEVA, BioBricks and Type IIS restriction enzyme standards," Microbial biotechnology, vol. 13, no. 6, pp. 1793–1806, 2020, doi: 10.1111/1751-7915.13609.
[8] E. Martínez-García et al., "SEVA 4.0: an update of the Standard European Vector Architecture database for advanced analysis and programming of bacterial phenotypes," Nucleic acids research, vol. 51, D1, D1558-D1567, 2023, doi: 10.1093/nar/gkac1059.
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