Composite

Part:BBa_K4586034

Designed by: Mohamed Mohamed Saad Aboelghare   Group: iGEM23_AFCM-Egypt   (2023-10-11)
Revision as of 14:07, 12 October 2023 by Ahmed Mattar (Talk | contribs) (→‎Experimental Characterization)

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Anti CD19 Syn-notch receptor

Usage and Description

This composite part codes for a synthetic receptor sensitive to the CD19 expressed on B-cells. Syn notch receptor consists of three main domains, first the extracellular domain represented in tagged citrullinated vimentin conjugated to CD8 alpha signal that mediates the expression of the receptor on the cell membrane, second the transmembrane domain represented in mouse notch core protein that is characterized by mechanosensitive structure that transfer the signal from the extracellular domain to the third intracellular domain that contains our potent transcription module VP64 which trigger the expression of our therapeutic agent (Cas12k\gBAFF-R) as shown in figure 1

Experimental Characterization

In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part present (p1) including (cd8-alpha,histag,zf21..16/vp64 ,and anti-CD19),and then we measured the specific concentration of the running part using Real-Time PCR as shown in the following figure.





We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure lane(P1).



After the ligation step, we did culture of the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the plasmid. The cell culture plate of transformed pCDNA vector containing insert parts is shown in the following figure. This plasmid contains 1-Syn-notch (CD8 alpha-his tag-Anti CD19-mouse notch core-ZF21.16\VP64)) 2-Booster gene 1 (SDC4, STEAP3) 3-Booster gene 2 (NAdB)

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 614
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1174
    Illegal SapI.rc site found at 2056


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