Part:BBa_K4370010
STREPTO_E.coli_CLONING_MODULE
The part allows the easy cloning of Streptomyces genes under the control of a strong and constitutive promoter using a red/white screening. Therefore the module contains parts specific to Streptomyces chassis as well as parts dedicated to the expression in E. coli to perform cloning.
Notably, this part contains the Streptomyces constitutive strong promoter KasOP* (BBa_K4370006) followed by a RBS from of capsid protein obtained from Streptomyces temperate phage ϕC31 (BBa_K4370007) that has been previously described as highly efficient to promote translation when combined with the kasOP* promoter (Bai et al., PNAS, 2015). The part also contains a cassette for the expression of mRFP1 (BBa_K4370008) under the control of an E. coli promoter (BBa_K1155000) and an RBS (BBa_B0034). This RFP expression module is followed by two stop codons and two terminators (BBa_B0010 and BBa_B0012).
This cassette is meant to be replaced by the construction of interest and offers an easy means of visual screening the clones (pink if the E. coli cassette is still present in the plasmid i.e. if the clone contains the parental plasmid, white if not).
Usage and Biology
Streptomyces are chassis of great biotechnological interest to produce antibiotics but also antitumors, anti-cancer drugs, immunosuppressive agents, etc. Their genome is linear and highly rich in GC (circa 72 %). Their genetic modification generally relies on the introduction of plasmids by conjugation from E. coli to Streptomyces. It is therefore necessary to develop specific vectors (conjugative, GC rich, specific promoters) in order to use Streptomyces as chassis.
In 2019, Aubry and collaborators published a set of 12 standardized and modular vectors (pOSV801 to pOSV812) designed to integrate genes of interest at specific loci in Streptomyces genomes (Table 1).
These vectors are composed of 5 modules: i) the replication module, ii) the antibiotic resistance cassette module (apramycin, hygromycin or kanamycin resistance), iii) the origin of transfer module, iv) the integration module (into PhiBT1, PhiC31, pSAM2 or VWB integration sites), and v) the cloning module. In this collection, this latter does not contain any Streptomyces promoter.
In order to facilitate the cloning of genes of interest for strong and constitutive expression in Streptomyces, we have constructed a new biobrick that corresponds to a new cloning module for this collection of vectors. It can be introduced between NotI sites to replace the original module by an expression cassette harboring a strong and constitutive promoter active in Streptomyces (kasOP*, BBa_K4370006) as well as an RBS (BBa_K4370007). We have successfully introduced this module in pOSV805 vector (hygromycin resistance, integration at PhiBT1 site) (Figure 1).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 792
Illegal AgeI site found at 904 - 1000COMPATIBLE WITH RFC[1000]
References
Céline Aubry, Jean-Luc Pernodet , Sylvie Lautru, « Modular and Integrative Vectors for Synthetic Biology Applications in Streptomyces spp”, Appl Environ Microbiol, 2019 Aug 1;85(16):e00485-19 https://doi.org/10.1128/AEM.00485-19
Chaoxian Bai , Yang Zhang , Xuejin Zhao, Yiling Hu, Sihai Xiang, Jin Miao, Chunbo Lou, Lixin Zhang “Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces”, Proc Natl Acad Sci U S A , 2015 Sep 29;112(39):12181-6. https://www.pnas.org/doi/10.1073/pnas.1511027112
Weishan Wang, Xiao Li, Juan Wang, Sihai Xiang, Xiaozhou Feng, Keqian Yang, “An engineered strong promoter for Streptomycetes”, Appl Environ Microbiol., 2013 Jul;79(14):4484-92. https://doi.org/10.1128/AEM.00985-13
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