Part:BBa_K3341003
+36GFP
The nucleotide sequence of GFP (+ 36) was biosynthesis of Qingke, using PUC57 with ampicillin resistance. The N-terminal restriction site was KPN I, the C-terminal site was hind III, and the host cell was E.coli Top 10. GFP green fluorescent protein (GFP) is improved by adding 36 positive charges, making it easier to combine with negatively charged proteins through electrostatic force. Because GFP has intrinsic green fluorescence, it is easier to be located in cells and tissues, not only can determine the distribution of negatively charged proteins, but also, to a certain extent, can indicate the electrical properties and structural changes of binding proteins. Positively charged green fluorescent protein (GFP) provides negatively charged molecules with a way to change their electrical properties, bind to negatively charged molecules and label negative proteins.
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]
iGEM2020_Nanjing-China Experiment
After comparison and verification, the synthesized nucleotide sequence is translated into protein, which is consistent with the expected sequence. We ligated the +36GFP protein target gene sequence to the PET29a vector to obtain the recombinant plasmid PET29a-+36GFP. The recombinant plasmid was transformed into the competent cell BL21 (DE3), and the final concentration of 1mM IPTG was induced at 16°C overnight for subsequent protein purification. In the experiment of purifying +36GFP protein, we first use Ni column to purify the target protein with histidine tag, and then use ion exchange column to purify +36GFP protein (to achieve the purification of positively charged protein). The SDS-PAGE detection results of the protein purified by the above two columns are shown in the figure:
| None |