Difference between revisions of "Part:BBa K4165011"
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===Wet-Lab Results===
 
===Wet-Lab Results===
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UBE2N enzyme is a E2 ubiquitin-conjugating enzyme that has the ability to recruit the ubiquitin-protesome pathway, it was expressed in BL21 (DE3) 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 UBE2N in BL-21 using pGS-21a vector </p>
 
<p style=" font-weight: bold; font-size:14px;"> Transformation of His UBE2N in BL-21 using pGS-21a vector </p>
 
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/ube2n-pgs.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/ube2n-pgs.jpg" style="margin-left:200px;" alt="" width="500" /></p>
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Transformation was done using TSS protocol after testing different transformation protocols and optimizing the best conditions for the used protocol with a transformation efficiency = 96000 no. of transformants/ug and CFU/ml = 288000
 
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                                   Figure 2. Transformed plate of His UBE2N + pGS-21a
 
                                   Figure 2. Transformed plate of His UBE2N + pGS-21a
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/ube2n-pjet.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/ube2n-pjet.jpg" style="margin-left:200px;" alt="" width="500" /></p>
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Transformation was done using TSS protocol after testing different transformation protocols and optimizing the best conditions for the used protocol with a transformation efficiency = 180000 no. of transformants/ug and CFU/ml = 1152000
 
</html>
 
</html>
 
                                     Figure 3. Transformed plate of His UBE2N + pJET
 
                                     Figure 3. Transformed plate of His UBE2N + pJET
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<p><img src="https://static.igem.wiki/teams/4165/wiki/wet-lab/characterization/whatsapp-image-2022-10-12-at-5-22-43-pm-1.jpeg" style="margin-left:200px;" alt="" width="500" /></p>
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===

Revision as of 15:48, 12 October 2022


UBE2N

Ubiquitin-conjugating E2 ligase that has a role in the ubiquitination cascade for protein degradation.

Usage and Biology

This part encodes for the UBE2N enzyme which is a member of the E2 ubiquitin-conjugating enzyme family. It acts as the second enzyme in the ubiquitination cascade to receive the thio-esterified ubiquitin from the E1 active site. The cascade starts with UBE2W being one of the few E2 types which binds to Trim21 (E3 ligase) and this specificity for trim21 is due to the presence of RING domain. The next step is the formation of UBE2N/UBE2V2 complex which function in the chain building and extending poly-ubiquitin chains from the mono-ubiquitinated proteins by UBE2W. This protein may also be involved in DNA post replication repair according to studies that have been done on mice.


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 221
  • 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) 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.

Wet-Lab Results

UBE2N enzyme is a E2 ubiquitin-conjugating enzyme that has the ability to recruit the ubiquitin-protesome pathway, it was expressed in BL21 (DE3) to be used in in-vitro ubiquitination assay to proof the concept that our system recruits the 26S protesomal-ubiquitin cascade.

Transformation of His UBE2N in BL-21 using pGS-21a vector

Transformation was done using TSS protocol after testing different transformation protocols and optimizing the best conditions for the used protocol with a transformation efficiency = 96000 no. of transformants/ug and CFU/ml = 288000 

                                 Figure 2. Transformed plate of His UBE2N + pGS-21a

Transformation of His UBE2N in DH-5 alpha using pJET vector

Transformation was done using TSS protocol after testing different transformation protocols and optimizing the best conditions for the used protocol with a transformation efficiency = 180000 no. of transformants/ug and CFU/ml = 1152000 

                                   Figure 3. Transformed plate of His UBE2N + pJET

<img src="whatsapp-image-2022-10-12-at-5-22-43-pm-1.jpeg" style="margin-left:200px;" alt="" width="500" />

Refrences

1. UBE2N ubiquitin conjugating enzyme E2 N [Homo sapiens (human)] - Gene - NCBI. (2022). 2. Pharos: Ubiquitin-conjugating enzyme E2 N (Tchem). (2022). 3. Kleiger, G., & Mayor, T. (2014). Perilous journey: a tour of the ubiquitin-proteasome system. Trends in cell biology, 24(6), 352-359. 3. 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.