Coding

Part:BBa_K4497020

Designed by: Till Gundlach   Group: iGEM22_Munich   (2022-10-09)
Revision as of 13:40, 13 October 2022 by Till Gundlach (Talk | contribs)


MESA mCherrynb-L1-TEV


This part is one of eight receptor parts that can be used for the the MESA receptor system of the iGEM Team Munich 2022 [1]. The receptors are made of a nanobody receptor for either GFP or mCherry, followed by two different link lengths, and an intracellular domain of either the transcription factor tTA or the protease TEV. tTA is cut off the receptor on contact with the TEV protease. The release of the transcription factor can be used to induce reporters or other expression in the cell of interest.

Design & Cloning

Design

This part specifically is made of the following components:

  • mCherry nanobody receptor (Part:BBa_K4497014)
    • IL-11 Signaling Sequence (Part:BBa_K4497003): Signaling Sequence for Cell surface expression
    • Flag-tag (Part:BBa_K4497012): Flag epitope tag that enables surface expression detection using immunofluorescence labeling
    • HA-tag (Part:BBa_K1150016): HA epitope tag that enables surface expression detection using immunofluorescence labeling
    • Anti-mCherry nanobody (Part:BBa_K4497002): a nanobody capable of binding mCherry
  • Membrane linker 1(Part:BBa_K4497015): A short linker between the nanobody and transmembrane domain
  • TEV protease MESA domain (Part:BBa_K4497011)
    • CD 28 transmembrane domain (Part:BBa_K4497004): transmembrane anchor for the MESA system
    • Tobacco Etch Virus (TEV) Protease (Part:BBa_K4497005): Protease cleaving proteins the TEV recognition site

Cloning

We created this part using restriction cloning. The backbone pcDNA3.4 with the GFP nanobody receptor sequence was created by digesting the plasmid pcDNA3.1-SP-Flag HA-Tag-mCherrynb-gp130 using XbaI, EcoRI. The plasmid was kindly provided to us from Prof. Scheller [1].

The insert containing the membrane linker and tTA signaling MESA domain was created by PCR on the plasmid V2-MESA-35F-Tev (Addgene #84503 [2]). The PCR product was digested using XbaI, EcoRI before ligation and transformation into E.coli.

  • Primer F: TAAGCAGAATTCTCCGGCGGGAATCTGGTCGTGGTTGCTGGAG
  • Primer R: TAAGCATCTAGATTACTTGTACAGCTCGTCCATG

MESA System

Please find an in-depth analysis of the usage of this part at the page of MESA GFPnb-L1-tTA: Part:BBa_K4497017

These are all the MESA parts:

Usage and Biology

We used this part in mammalian cell culture: HEK293T, Cos7 in a S1 safety level lab.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 511
    Illegal SpeI site found at 976
    Illegal PstI site found at 373
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 511
    Illegal SpeI site found at 976
    Illegal PstI site found at 373
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 511
    Illegal BamHI site found at 94
    Illegal BamHI site found at 127
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 511
    Illegal SpeI site found at 976
    Illegal PstI site found at 373
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 511
    Illegal SpeI site found at 976
    Illegal PstI site found at 373
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None