Difference between revisions of "Part:BBa K4165011"

Latest revision as of 18:48, 13 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

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 an E2 ubiquitin-conjugating enzyme that has the ability to recruit the ubiquitin-proteasome pathway, it was cloned in DH-5 alpha using a pJET cloning vector. Then we extract the plasmid and restrict the UBE2N gene to ligate it with pGS-21a and transform them to be expressed in BL21 to be used in an in-vitro ubiquitination assay to prove the concept that our system recruits the 26S proteasomal-ubiquitin cascade.

Ligation reaction between Ube2N and pJET cloning vector

We used t4 ligase to ligate Ube2N with pJET cloning vector so, we incubated Ube2N with pJET overnight at 15°C.

     Figure 2. This figure shows the ligation reaction between Ube2N and pJET cloning vector, UBE2N part size is 625 
      bp so the band is expected to appear between 500 bp and 750 bp as shown in the gel.

Transformation of His UBE2N 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 three 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 was calculated for Ube2N in pGS-21a expression vector and in pJET cloning vector and they were found to be = 96000 no. of transformants/ug and 180000 no. of transformants/ug respectively

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

                                   Figure 3. Transformed plate of His UBE2N + pJET

                                 Figure 4. Transformed plate of His UBE2N + pGS-21a

Affinity chromatography of UBE2N

Affinity chromatography is a technique used to purify the proteins to get a pure protein alone without the cell lysate so, we performed affinity chromatography for total protein extraction to get the UBE2N alone.

           Figure 5. This figure shows the BCA assay results of affinity chromatography after protein extraction.

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.