Difference between revisions of "Part:BBa K4165015"
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===Usage and Biology===
 
===Usage and Biology===
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Inactive RING/U-box type E3s, such lacking the active site cysteine residues to form thioester linkages with ubiquitin, or even the absence of E3 will not prevent UBE2W-catalyzed ubiquitination, albeit at a slow rate.
 
  
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===source===
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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 the 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.
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UBE2W: Q96B02 in Uniprot - NP_001001481.3 in NCBI
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===<span class='h3bb'>Sequence and Features</span>===
 
===<span class='h3bb'>Sequence and Features</span>===
 
<partinfo>BBa_K4165015 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4165015 SequenceAndFeatures</partinfo>
  
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===Dry Lab===
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<p style=" font-weight: bold; font-size:14px;"> Mathematical modeling </p>
 +
<p style=" font-weight: bold; font-size:14px;">Transcription rate and translation rate under T7 promotor </p>
 +
the mathematical modeling was based on our code for the calculation of transcription and translation (you can find it in the code section) besides the estimated results from the wet lab.
 +
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/dry-lab/mathematical-modeling/be2wu2.png" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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                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 an E2 ubiquitin-conjugating enzyme that has the ability to recruit the ubiquitin-proteasome pathway. In the wet lab, we cloned it in DH-5 alpha using a pJET cloning vector then we did manual plasmid miniprep to extract the part, and ligate it to be expressed in BL-21 using a pGS-21a expression vector to used in in-vitro ubiquitination assay to prove the concept that our system recruits the 26S proteasomal-ubiquitin cascade.
 +
<p style=" font-weight: bold; font-size:14px;"> Transformation of His UBE2W in BL-21 using pGS-21a expression vector and in DH-5 alpha using pJET cloning vector</p>
 +
The transformation was done using the TSS protocol after testing different buffers which are TSS buffer, Calcium Chloride, and a combination between Calcium Chloride and Magnesium Chloride. We optimized our protocol to use the TSS buffer as it showed the best results. The transformation efficiency and CFU/ml were calculated for UBE2W in the pGS-21a expression vector and in the 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.
 +
 +
<p style=" font-weight: bold; font-size:14px;"> Transformation of His UBE2W in DH-5 alpha using pJET cloning vector </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>
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                                  Figure 2. Transformed plate of His UBE2W + pJET
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<html>
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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>
 +
</html>
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                                Figure 3. Transformed plate of His UBE2W + pGS-21a
 +
<p style=" font-weight: bold; font-size:14px;"> Affinity chromatography of Ube2W </p>
 +
Affinity chromatography is a technique used to purify the proteins to get the protein alone without the cell lysate. So, we performed affinity chromatography for total protein extraction to get pure UBE2W.
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/standard-curve.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
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<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/ube2w.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
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        Figure 4. This figure shows the BCA assay results of the affinity chromatography that was done after the protein
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                                                            extraction.
  
 
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===Refrences===
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===References===
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1- Komander D. (2009). The emerging complexity of protein ubiquitination. Biochemical Society transactions, 37(Pt 5), 937–953. https://doi.org/10.1042/BST0370937
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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.
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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.
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2- David, Y., Ziv, T., Admon, A., & Navon, A. (2010). The E2 ubiquitin-conjugating enzymes direct polyubiquitination to preferred lysines. The Journal of biological chemistry, 285(12), 8595–8604. https://doi.org/10.1074/jbc.M109.089003
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Latest revision as of 22:49, 13 October 2022


UBE2W

Ubiquitin-conjugating E2 ligase 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 the 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


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
    COMPATIBLE 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) besides 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 an E2 ubiquitin-conjugating enzyme that has the ability to recruit the ubiquitin-proteasome pathway. In the wet lab, we cloned it in DH-5 alpha using a pJET cloning vector then we did manual plasmid miniprep to extract the part, and ligate it to be expressed in BL-21 using a pGS-21a expression vector to used in in-vitro ubiquitination assay to prove the concept that our system recruits the 26S proteasomal-ubiquitin cascade.

Transformation of His UBE2W in BL-21 using pGS-21a expression vector and in DH-5 alpha using pJET cloning vector

The transformation was done using the TSS protocol after testing different buffers which are TSS buffer, Calcium Chloride, and a combination between Calcium Chloride and Magnesium Chloride. We optimized our protocol to use the TSS buffer as it showed the best results. The transformation efficiency and CFU/ml were calculated for UBE2W in the pGS-21a expression vector and in the 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.

Transformation of His UBE2W in DH-5 alpha using pJET cloning vector 

                                 Figure 2. Transformed plate of His UBE2W + pJET 



                               Figure 3. Transformed plate of His UBE2W + pGS-21a

Affinity chromatography of Ube2W

Affinity chromatography is a technique used to purify the proteins to get the protein alone without the cell lysate. So, we performed affinity chromatography for total protein extraction to get pure UBE2W. 

       Figure 4. This figure shows the BCA assay results of the affinity chromatography that was done after the protein 
                                                            extraction.


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.