Difference between revisions of "Team:UST Beijing/Pangu"

Pangu

1w6k and Ginseng Cyclase

As shown in Figure 1, the human OSC (also namely lanosterol synthesis, LAS), catalyzes the 2,3-oxidosqualene to form lanosterol via cyclization (to yield a key intermediate: protosterol cation), rearrangement, hydrogen transfer, methyl transfer, and dehydro-condensation. Lanosterol is the first sterol to be formed in the MVP and the essential intermediate to form cholesterol. This extraordinary reaction has been described as the most complicated enzyme catalytic reaction in human biology. There are two available crystal structures of human OSC (PDB ID: 1w6k and 1w6j): one contains a inhibitor RO48−8071 (1w6j) and the other contains the lanosterol (1w6k).

Plasmid Design

We used Swiss Model making the three-Dimension model of ginseng cyclase. And then we used the Chimera match amino acid sequences and constructions of 1w6k(the human cyclase) and ginseng cyclase. We found the first 90 amino acids on the two sequence was extremely different, which indicates obvious species specificity. In order to allow the squalene cyclase can function in the human body later, we replaced the 100 amino acid residues on N-terminal of the ginseng cyclase with 90 amino acid residues on N-terminal of 1w6k. And we found there are 5 amino acid residues site existing steric to the new peptide, so we switched them to the amino acid residues of same site on 1w6k.

We used the geneart to do reverse translation to get the nucleotide sequence of pangu cyclase and the website continue optimization codes. Finally we added the other ending codes to gene sequence. Because the optimal gene fragment that the Gene synthesis company can synthesize is 1600bp, so we cut target fragment of 2224bp into two sections on average.

Plasmid Construction(BBa_K2519000)

At first, we used the Gibson Assembly Master Mix to assemble the target fragments and pSB1c3 backbone.

Incubate at 50℃ for 1 hour. But we got many results of false positive. There appeared white and pink bacterial colonies on our medium plate.

And the SDS-agarose gel electrophoresis figure only showed the electrophoresis strips about 2000bp and 3000bp, which are backbone and plasmid containing red fluorescent gene. And we have tried many times, but we can’t find any colony containing our designed plasmid.

Finally we used PCR to amplificate the DNA fragments and isolated the DNA sequence of 2224bp. The target segment and the plasmid backbone of pSB1C3 were digested by the PstI ang EcoRI enzyme respectively. Then we used T4 DNA ligase to connect the backbone and target fragment.

Incubate at 16℃ for 2 hours.

The electrophoresis figure2 showed our designed plasmid.

Because not every team has the Dpn1 enzyme to cut up original template DNA. So we want to make a suggestion for IGEM foundation, the community may use Dpn1 enzyme to cut up the RFP plasmid before delivery of backbone.

Plasimd Sequencing

Comparing the sequencing result with the sequence of pangu cyclase, we found there were two mutation sites on the sequence.

According to the figure1, AAG mutated to GAG, so the 30th amino acid on peptide mutated from Arginine to glutamic acid.

As is showed on figuer2, ATC mutate to ATA, but the amino acid keep as Isoleucine.

The first site may produce influence for the expression of squalene cyclase, which we have not known. We will verificate the function of the part later.