Coding

Part:BBa_K4768000

Designed by: BOABEKOA Pakindame   Group: iGEM23_Toulouse-INSA-UPS   (2023-09-12)
Revision as of 16:13, 20 September 2023 by Clea19 (Talk | contribs)


DhdR repressor

Part for expression and purification of our transcriptional repressor DhdR for biosensing.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 47
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 493
    Illegal BglII site found at 622
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 756


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Introduction

The CALIPSO part (BBa_K4768000) consists of the transcriptional repression factor, DhdR, which has been isolated from the bacterium Achromobacter denitrificans. The codon sequence has been optimized for expression in E.coli. Additionally, the presence of a T7 promoter and terminator enables its inducible expression by IPTG in E.coli BL21 (DE3). Finally, a Histidine tag is included in the sequence to facilitate the purification of DhdR.

This transcriptional repressor was employed in our biosensing system, which was inspired by the work of Ping Xu et al. 1 and the work conducted by iGEM Duke 2021. Our objective was to use the affinity between 2-Hydroxyglutarate, an oncometabolite, and DhdR to trigger the production of our drug-activating enzyme when liposomes are situated in a tumoral environment. To achieve this, we positioned our gene of interest under the control of the operator site of DhdR, referred to as dhdO.

<b>Figure 1: Functioning of our biosensor.</b> In presence of 2-HG, the repression is removed and the gene is expressed leading to the production of the thymidine phosphorylase which converts our Tegafur prodrug into an active anticancer drug, 5-fluorouracile.Figure 1: Functioning of our biosensor. In presence of 2-HG, the repression is removed and the gene is expressed leading to the production of the thymidine phosphorylase which converts our Tegafur prodrug into an active anticancer drug, 5-fluorouracile.

Construction

The CALIPSO part BBa_K4768000 comprises the transcriptional repression factor DhdR fused with a histidine tag at its N-terminus. The synthesis of this gBlock was performed and provided by IDT.

The gBlock was then cloned into the pET_21a(+) plasmid and transformed into Stellar competent cells.

Primers used to clone this part in the pET21: (from 5' to 3'):

Molecular Modeling

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Characterisation

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Conclusion and Perspectives

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References

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  2. article 2 xxxxxxx
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