Difference between revisions of "Part:BBa K4165015"
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===WetLab Results=== | ===WetLab Results=== | ||
| − | UBE2W enzyme is a E2 ubiquitin-conjugating enzyme that has the ability to recruit the ubiquitin-protesome pathway, it was expressed in | + | UBE2W enzyme is a E2 ubiquitin-conjugating enzyme that has the ability to recruit the ubiquitin-protesome pathway, it was cloned in DH-5 alpha using pJET cloning vector then it was expressed in BL-21 using pGS-21a expression vector to be used in in-vitro ubiquitination assay to proof the concept that our system recruits the 26S protesomal-ubiquitin cascade. |
| − | <p style=" font-weight: bold; font-size:14px;"> Transformation of His UBE 2w in BL-21 using pGS-21a vector </p> | + | <p style=" font-weight: bold; font-size:14px;"> Transformation of His UBE 2w in BL-21 using pGS-21a expression vector and in DH-5 alpha using pJET cloning vector</p> |
| − | Transformation was done using TSS protocol after testing different | + | Transformation was done using TSS protocol after testing different buffers which are: TSS buffer, Calcium Chloride and combination between Calcium Chloride and Magnesium Chloride we optimized our protocol to use TSS buffer as it showed the best results. Transformation effeciency and CFU/ml were calculated for Ube2W in pGS-21a expression vector and in pJET cloning vector and they were found to be 576000 no. of transformants/ug and CFU/ml = 1152000 and 240000 no. of transformants/ug and CFU/ml = 480000 respectively |
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/ube2w-pgs.jpg" style="margin-left:200px;" alt="" width="500" /></p> | <p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/ube2w-pgs.jpg" style="margin-left:200px;" alt="" width="500" /></p> | ||
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Figure 2. Transformed plate of His UBE 2w + pGS-21a | Figure 2. Transformed plate of His UBE 2w + pGS-21a | ||
<p style=" font-weight: bold; font-size:14px;"> Transformation of His UBE 2w in DH-5 alpha using pJET vector </p> | <p style=" font-weight: bold; font-size:14px;"> Transformation of His UBE 2w in DH-5 alpha using pJET vector </p> | ||
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/ube2w-pjet.jpg" style="margin-left:200px;" alt="" width="500" /></p></html> | <p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/ube2w-pjet.jpg" style="margin-left:200px;" alt="" width="500" /></p></html> | ||
Figure 3. Transformed plate of His UBE 2w + pJET | Figure 3. Transformed plate of His UBE 2w + pJET | ||
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<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
Revision as of 09:14, 13 October 2022
UBE2W
Ubiquitin-conjugating E2 ligase that has a role in the ubiquitination cascade for protein degradation.
Usage and Biology
This E2 ubiquitin-conjugating enzyme UBE2W is the key participant in the set of enzymes as it is responsible for the initial step of monoubiquitylation by TRIM21 E3 ligase. The UBE2W is most specific for RING domain E3 ligases which happens to be that Trim21, which we are working with, is one of those RING domain E3 ligases.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Dry Lab
Mathematical modeling
Transcription rate and translation rate under T7 promotor
the mathematical modeling was based on our code for the calculation of transcription and translation (you can find it in the code section) beside with the estimated results from the wet lab.
Figure 1. this figure shows the results from the transcription and translation code showing the
variation of mRNA and protein concentrations with time compared with the wet lab results.
WetLab Results
UBE2W enzyme is a E2 ubiquitin-conjugating enzyme that has the ability to recruit the ubiquitin-protesome pathway, it was cloned in DH-5 alpha using pJET cloning vector then it was expressed in BL-21 using pGS-21a expression vector to be used in in-vitro ubiquitination assay to proof the concept that our system recruits the 26S protesomal-ubiquitin cascade.
Transformation of His UBE 2w in BL-21 using pGS-21a expression vector and in DH-5 alpha using pJET cloning vector
Transformation was done using TSS protocol after testing different buffers which are: TSS buffer, Calcium Chloride and combination between Calcium Chloride and Magnesium Chloride we optimized our protocol to use TSS buffer as it showed the best results. Transformation effeciency and CFU/ml were calculated for Ube2W in pGS-21a expression vector and in pJET cloning vector and they were found to be 576000 no. of transformants/ug and CFU/ml = 1152000 and 240000 no. of transformants/ug and CFU/ml = 480000 respectively
Figure 2. Transformed plate of His UBE 2w + pGS-21a
Transformation of His UBE 2w in DH-5 alpha using pJET vector
Figure 3. Transformed plate of His UBE 2w + pJET
References
1. Stewart, M. D., Ritterhoff, T., Klevit, R. E., & Brzovic, P. S. (2016). E2 enzymes: more than just middle men. Cell research, 26(4), 423-440. 2. Vittal, V., Wenzel, D. M., Brzovic, P. S., & Klevit, R. E. (2013). Biochemical and structural characterization of the ubiquitin-conjugating enzyme UBE2W reveals the formation of a noncovalent homodimer. Cell biochemistry and biophysics, 67(1), 103-110.