Difference between revisions of "Part:BBa K4586018"
Ahmed Mattar (Talk | contribs) (→Literature Characterization) |
Ahmed Mattar (Talk | contribs) (→Experimental Characterization) |
||
| (9 intermediate revisions by 2 users not shown) | |||
| Line 14: | Line 14: | ||
position: relative; | position: relative; | ||
top: 50%; | top: 50%; | ||
| − | left: | + | left: 25%; |
transform: translate( -50%); | transform: translate( -50%); | ||
padding-bottom:25px; | padding-bottom:25px; | ||
| Line 28: | Line 28: | ||
==Experimental Characterization== | ==Experimental Characterization== | ||
| − | In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents in lane (P1) including CD8 alpha-his tag-mouse notch core-ZF21.16\VP64, and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure. | + | In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents in lane (P1) including CD8 alpha-his tag-mouse notch core-ZF21.16\VP64, and anti-CD19 and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure. |
<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style=" max-width:850px; | <html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style=" max-width:850px; | ||
width:100%; | width:100%; | ||
| Line 55: | Line 55: | ||
padding-top:25px; | padding-top:25px; | ||
"src="https://static.igem.wiki/teams/4586/wiki/parts-experiments/digestion-2.png"> | "src="https://static.igem.wiki/teams/4586/wiki/parts-experiments/digestion-2.png"> | ||
| + | <p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span | ||
| + | lang=EN style='font-size:11.0pt;line-height:115%'> | ||
| + | |||
| + | </span></p></div></html> | ||
| + | <br><br> | ||
| + | After the ligation step, we did culture of the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the plasmid. | ||
| + | The cell culture plate of transformed pCDNA vector containing insert parts is shown in the following figure. | ||
| + | This plasmid contains | ||
| + | 1-Syn-notch (CD8 alpha-his tag-Anti CD19-mouse notch core-ZF21.16\VP64)) | ||
| + | 2-Booster gene 1 (SDC4, STEAP3) | ||
| + | 3-Booster gene 2 (NAdB) | ||
| + | <br> | ||
| + | <html><div align="center"style="border:solid #17252A; width:75%;float:center;"><img style=" max-width:850px; | ||
| + | width:75%; | ||
| + | height:auto; | ||
| + | position: relative; | ||
| + | top: 50%; | ||
| + | left: 35%; | ||
| + | transform: translate( -50%); | ||
| + | padding-bottom:25px; | ||
| + | padding-top:25px; | ||
| + | "src="https://static.igem.wiki/teams/4586/wiki/parts/vector-1.png"> | ||
<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span | <p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span | ||
lang=EN style='font-size:11.0pt;line-height:115%'> | lang=EN style='font-size:11.0pt;line-height:115%'> | ||
Latest revision as of 14:01, 12 October 2023
CD 8 alpha signal
Description
CD8 alpha is signal peptide translocate the conjugated protein to the cellular membrane.
Literature Characterization
The study created a reporter construct by joining the C-terminus of CD63, one of the most used exosome markers, to nanoluc (nluc), a tiny and potent bioluminescence reporter10. After progressive centrifugation to eliminate masking signals12, luminescence in the cell-culture supernatant was measured. This reporter gene was co-transfected with plasmids expressing potential candidates for exosome production augmentation.
Poly(A) RNA samples from each cell line were separated by gel electrophoresis, transferred to a nitrocellulose membrane, and hybridized with 32P-labeled cDNA probes specific for MC-CPA or CD8amp;. The membranes were then reprobed with a 32P-labeled p-actin cDNA probe to verify that equal amounts of RNA had been loaded in each lane. The results showed that MC-CPA mRNA was expressed at a higher level in MW9.11-3 cells than in MC/9.IL-4 cells, while CDalpha mRNA was expressed at a similar level in both cell lines. These findings suggest that MC-CPA and CDalpha may play different roles in the development and differentiation of these two cell
Experimental Characterization
In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents in lane (P1) including CD8 alpha-his tag-mouse notch core-ZF21.16\VP64, and anti-CD19 and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure.
We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure in lane (P1)
After the ligation step, we did culture of the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the plasmid. The cell culture plate of transformed pCDNA vector containing insert parts is shown in the following figure. This plasmid contains 1-Syn-notch (CD8 alpha-his tag-Anti CD19-mouse notch core-ZF21.16\VP64)) 2-Booster gene 1 (SDC4, STEAP3) 3-Booster gene 2 (NAdB)
References
Hara, T., Harada, N., Mitsui, H., Miura, T., Ishizaka, T., & Miyajima, A. (1994). Characterization of cell phenotype by a novel cDNA library subtraction system: expression of CD8 alpha in a mast cell-derived interleukin-4-dependent cell line.
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]