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

Revision as of 01:48, 1 November 2017

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Pangu

1w6k and Ginseng Cyclase

Figure 1. Cholesterol biosynthetic pathway.

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).

Figure 2. Brief triterpenoid biosynthetic pathway.

As seen from Figure 2, OSC enzymes are critical to sterol synthesis not only in human being but also in high plants and fungi. There are several OSC isoforms for both sterol synthesis and triterpenoid biosynthesis in the higher plants, such as PNA, bAS, and so on. After constructing the basic cyclic triterpenoid skeletons by OSC, these skeletons can form various cyclic terpene derivatives with oxidation and glycosylation. This concerted reaction also existed in another squalene cyclase, squalene-hopene cyclase (SHC). It can be seen that the product of human cyclase is Lanosterol ,and the product of ginseng cyclase is dammarenediol-II,the difference lies in the spatial structure, the position of the hydroxyl and Carbon-carbon double bonds is different. It can be seen that the two kinds of enzymes react with the same reactants, but the product is different in spatial structure and bond position.

We put the ginseng cyclase sequence into NCBI, compared with the sequence of similar species, save the alignment results, into the CLC, and then the human cyclase sequence is also introduced into the CLC, and made a phylogenetic trees. From the phylogenetic trees we can see that ginseng cyclase and human cyclase are far apart. It shows that their sequence are difference. Crystal structure of their primary also have a marked difference. According to Pangu algorithm and literature, we choose people cyclase 1w6k and ginseng cyclase combined, we obtain the required Pangu cyclase.

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.

Sequence1（25ng/μL）	4μL
Sequence2（25ng/μL）	4μL
pSB1C3 backbone（25ng/μL）	2μL
Enzyme	10μL
Total	20μL

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.

Target fragment（40ng/μL）	4μL
Backbone（17ng/μL）	3μL
10Xbuffer	2μL
T4 DNA ligasee	0.4μL
ddH2O	11μL
Total	20.4μL

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.