Difference between revisions of "Part:BBa K5036001"

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lang=EN style='font-size:11.0pt;line-height:115%'>Figure A shows how the concentration of wild-type TEV protein compares to the L56V variant. A plateau in the absorption reading indicates the point at which the protein precipitates out of solution (limited solubility). Introducing mutations L56V and S135G significantly increased TEV solubility (Figure B), allowing them to reach maximum concentrations of 6.2 mg/mL and 5.77 mg/mL, respectively. Since the remaining variants (K45F, Q58F, E106G) displayed similar solubility to the wild-type TEV, they were not investigated further.
 
lang=EN style='font-size:11.0pt;line-height:115%'>Figure A shows how the concentration of wild-type TEV protein compares to the L56V variant. A plateau in the absorption reading indicates the point at which the protein precipitates out of solution (limited solubility). Introducing mutations L56V and S135G significantly increased TEV solubility (Figure B), allowing them to reach maximum concentrations of 6.2 mg/mL and 5.77 mg/mL, respectively. Since the remaining variants (K45F, Q58F, E106G) displayed similar solubility to the wild-type TEV, they were not investigated further.
 
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==Software Characterization==
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To illustrate TEV protease assembly, we used alpha fold 3 to show the interactions between its C and N domains.
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<div align="center"style="border:solid #17252A; width:100%;float:center;">
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<img  style="width:25vw;" src="https://static.igem.wiki/teams/5036/part-software/alignment-plot-tev-protease.png" alt="" />
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            <h3 class="fade-in">Alignment Plot</h3>
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            <img  style="width:25vw" src="https://static.igem.wiki/teams/5036/part-software/c-ntevannotated-ezgif-com-video-to-gif-converter.gif" alt="" />
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            <h3 class="fade-in">3D structure of TEV protease</h3>
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='padding-bottom:40px;font-size:11.0pt;line-height:115%'>The alignment plot reflects that the receptor’s 3D structure has positive alignment with the experimental structures used in alpha fold 3. The results indicate favorable protein structure
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==Reference==
 
==Reference==
 
Cabrita, L. D., Gilis, D., Robertson, A. L., Dehouck, Y., Rooman, M., & Bottomley, S. P. (2007). Enhancing the stability and solubility of TEV protease using in silico design. Protein science, 16(11), 2360-2367.‏
 
Cabrita, L. D., Gilis, D., Robertson, A. L., Dehouck, Y., Rooman, M., & Bottomley, S. P. (2007). Enhancing the stability and solubility of TEV protease using in silico design. Protein science, 16(11), 2360-2367.‏

Revision as of 05:09, 25 September 2024


C-TEV

Part Description

This is known as the tobacco etch virus which is very selective for cleaving proteins at particular amino acid sequences and has been modified to have better qualities like greater heat stability, decreased self-cleavage and also allows researchers to precisely cleave the tag off, leaving behind the receptor in its unmodified form. In our model, TEV was divided into N-terminal and C-terminal fragments. So the C-terminal fragment was grafted onto the first chain of our dCas9 synRTK receptor.

Usage

Our dCas9-synRTK receptor is activated when VEGF binds to it causing (c)-TEV to bind to (N)-TEV to produce TEV protease, which cleaves TCS (Q, G) and TCS (Q, L) sites to release dcas9 from the receptor.

This figure illustrates the structure of c-terminal domain of TEV in first chain of dCas9 synRTK receptor. .

Literature Characterization

This study investigated five mutations introduced into the TEV protease enzyme to improve its solubility. Following the mutations, a technique involving centrifugation was used to evaluate the solubility of each variant. The variants were concentrated ,samples were collected at specific time points, and then the protein concentrations were measured to assess their solubility.

Figure A shows how the concentration of wild-type TEV protein compares to the L56V variant. A plateau in the absorption reading indicates the point at which the protein precipitates out of solution (limited solubility). Introducing mutations L56V and S135G significantly increased TEV solubility (Figure B), allowing them to reach maximum concentrations of 6.2 mg/mL and 5.77 mg/mL, respectively. Since the remaining variants (K45F, Q58F, E106G) displayed similar solubility to the wild-type TEV, they were not investigated further.

Software Characterization

To illustrate TEV protease assembly, we used alpha fold 3 to show the interactions between its C and N domains.

Alignment Plot

3D structure of TEV protease

The alignment plot reflects that the receptor’s 3D structure has positive alignment with the experimental structures used in alpha fold 3. The results indicate favorable protein structure .

Reference

Cabrita, L. D., Gilis, D., Robertson, A. L., Dehouck, Y., Rooman, M., & Bottomley, S. P. (2007). Enhancing the stability and solubility of TEV protease using in silico design. Protein science, 16(11), 2360-2367.‏


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 310