Coding

Part:BBa_K3930026

Designed by: Thomas Gaudin   Group: iGEM21_Toulouse_INSA-UPS   (2021-10-08)
Revision as of 10:18, 15 October 2021 by Ambre-Jousselin (Talk | contribs)


3,6-nonadienal induction system and expression in S. elongatus (pCONCOMBRE) Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2244
    Illegal EcoRI site found at 2250
    Illegal XbaI site found at 590
    Illegal XbaI site found at 6286
    Illegal XbaI site found at 8003
    Illegal XbaI site found at 8030
    Illegal PstI site found at 8476
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2244
    Illegal EcoRI site found at 2250
    Illegal NheI site found at 777
    Illegal NheI site found at 872
    Illegal NheI site found at 899
    Illegal PstI site found at 8476
    Illegal NotI site found at 7951
    Illegal NotI site found at 7959
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2244
    Illegal EcoRI site found at 2250
    Illegal BglII site found at 6283
    Illegal BglII site found at 6289
    Illegal BamHI site found at 3717
    Illegal XhoI site found at 3753
    Illegal XhoI site found at 4736
    Illegal XhoI site found at 6361
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2244
    Illegal EcoRI site found at 2250
    Illegal XbaI site found at 590
    Illegal XbaI site found at 6286
    Illegal XbaI site found at 8003
    Illegal XbaI site found at 8030
    Illegal PstI site found at 8476
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2244
    Illegal EcoRI site found at 2250
    Illegal XbaI site found at 590
    Illegal XbaI site found at 6286
    Illegal XbaI site found at 8003
    Illegal XbaI site found at 8030
    Illegal PstI site found at 8476
    Illegal NgoMIV site found at 1936
    Illegal AgeI site found at 2163
    Illegal AgeI site found at 2190
    Illegal AgeI site found at 4955
    Illegal AgeI site found at 7844
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 8686
    Illegal BsaI.rc site found at 911
    Illegal SapI site found at 2653
    Illegal SapI site found at 3728
    Illegal SapI.rc site found at 7355

Introduction

The pCONCOMBRE part (BBa_K3930026) enables the production of 3,6-nonadienal from linolenic and linoleic acids, and is composed of:

- the RA (BBa_K3930027) and LA (BBa_K3930028) integration sites in the NSI locus of S.elongatus genome (based on the plasmid pAM4951 from Easyclone Marker free kit (Taton et al. 2014))

- the Nb-9-LOX (BBa_K3930030), Cm-9-HPL (BBa_K3930031) and LacI genes (Part:BBa_C0012), for the production of 3,6-nonadienal and LacI repressor. The sequences of the Nb-9-LOX and the Cm-9-HPL were codon optimized for expression into S.elongatus

- the IPTG and theophylline inducible promoter Trc-theoE-riboswitch (BBa_K3930029) , driving the expression of Nb-9-LOX and Cm-9-HPL

- the resistance marker SpecR (BBa_K3930032) to select for cyanobacteria integrants

Construction

IDT performed DNA synthesis and delivered the part as gBlock. The construct was cloned with an In-Fusion Takara kit into the pAM4951 plasmid and then transformed into E.coli Dh5α strain. Figure 1 shows the restriction map of the resulting clones. The expected restriction profile was obtained for clone A5.

Figure 1: pCONCOMBRE assembly

pCONCOMBRE restriction profile from clone A5 was checked by digestion visualised on EtBr stained agarose electrophoresis gel. A theoretical gel is presented on the right gel and the NEB 1 kb DNA ladder on the left (note that a different ladder is presented on the theoretical gel)


pCONCOMBRE insert was then linearized by inverse PCR. The amplicon was integrated into the genome of S.elongatus strain following the triparental conjugation protocol of Gale et al. (2019). Figure 2 shows the electrophoresis gel of colony PCR to verify integrants genotype. The expected sizes were obtained for clone 4, but one amplicon (LA) had not the right size, however, all genes from the insert were detected.


Figure 2: Integration of pCONCOMBRE in S.elongatus

pCONCOMBRE integration from clone D2 was checked by PCR visualised on EtBr stained agarose electrophoresis gel. A theoretical gel is presented on the right of and the NEB 1 kb DNA ladder on the left (note that a different ladder is presented on the theoretical gel)


pCONCOMBRE insert integration at NSI was nonetheless successful. The integrant strain was named Synecho-pCONCOMBRE and saved as glycerol stock.

Conclusion and Perspectives

The pCONCOMBRE insert was successfully integrated into the S.elongatus genome. Nonetheless, the characterisation of the production of the 3,6-nonadienal has not been conducted, future iGEM teams will have to perform their own tests to verify the functionality of the construct.

References

  1. Gale GAR, Osorio AAS, Puzorjov A, Wang B, McCormick AJ. 2019. Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit. JoVE (Journal of Visualized Experiments).(152):e60451. doi:10.3791/60451.
  2. Taton A, Unglaub F, Wright NE, Zeng WY, Paz-Yepes J, Brahamsha B, Palenik B, Peterson TC, Haerizadeh F, Golden SS, et al. 2014. Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria. Nucleic Acids Res. 42(17):e136. doi:10.1093/nar/gku673.
    3.




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