Team:ECUST China/Contribution

Site-directed mutation
Innovation methods in Modeling
Construction of 3D-hardware
Supplement of existing parts

Our project makes contribution to the research of site-directed mutation, Innovation methods in modeling, the construction of 3D-hardware and supplement of existing parts.

Site-directed mutation

In order to further improve the heat-resistant property of protein, we use site-directed mutation to improve heat stability of phycocyanin which can give iGEMers some guidence on how to use site-directed mutant on proteins to improve it’s stability or provide our mutated sequence to those teams that will use phycocyanin α-subunit (cpcA encodes) in the future. Also the site-directed mutation can contribute to guide the site-directed mutation experiment with theoretical support, guiding the experiment proceeding more fluently.

Give our gene CpcA as an example. CpcA gene encodes α-subunit of phycocyanin which is a light-harvesting photosynthetic bile pigment-protein from the phycobiliprotein complex (phycobilisome, PBS) and is sensitive to heat and light. Through mutate the cpc A, we found the key amino acid of increasing the heat stability.

Here are the mutation steps:

First of all, we compared the amino acid sequence of ordinary phycocyanin with that of heat-resistant phycocyanin to find out the difference between them in the sequence. [Aa that needs mutation is marked in blue (40 in total):]

5	10	15	20	25	30	35	40
MKTPI	TEAIA	AADTQ	GRFLS	NTELQ	AADGR	FKRAV	ASMEA
ARALT	NNAQS	LIDGA	AQAVY	QKFPY	TTTMQ	GSQYA	STPEG
KAKCA	RDIGY	YLRMV	TYCLV	AGGTG	PMDEY	LIAGL	AEINS
TFDLS	PSWYV	EALKY	IKANH	GLSGQ	AAVEA	NAYID	YAINA	LS

And then used I-Mutant website for operation. The following are the operation steps on the website:
1.

Protein sequence-----Enter
2.

[ Protein sequence ]:
MKTPITEAIAAADTQGRFLSNTELQAADGRFKRAVASMEAARALTNNAQSLIDGAAQAVYQKFPYTTTMQGSQYASTPEGKAKCARDIGYYLRMVTYCLVAGGTGPMDEYLIAGLAEINSTFDLSPSWYVEALKYIKANHGLSGQAAVEANAYIDYAINALS
[ Position ]: (For example, if the first AA to be mutated is "I", fill in "5")
[ New Residue ]: Fill in the original amino acid to be mutated
[ Temperature ]: 25
[ Ph ]: 7
[ Prediction ]: (DDG)
[ E-mail ]: (Fill in your email, the result will be sent to the email)

Negative DDG means that mutation improves stability. Three New Residue records with the largest absolute value of negative values are selected from 19 mutations, such as 5 I--H, T and Y (" 5 "is the position of mutation AA in the whole sequence, I is the original AA, and the absolute value of negative values of H, T and G decreases successively). Here are our results.

5	I--D-2.1	G-2.01	W-1.74
9	I--G-2.66	P-2.36	D-2.32
10	A--P-2.15	K-1.59	Y-1.56
11	A--P-1.80	K-1.53	Y-1.31
14	T--P-1.62	G-0.93	A-0.86
21	N--G-1.93	K-1.46	A-1.35
26	A--P-2.15	G-2.10	K-1.96
28	D--G-2.11	P-1.76	K-1.69
31	F--G-3.53	D-2.27	A-2.09
32	K--D-1.78	G-1.15	T-0.99
33	R--S-1.46	K-1.37	D-1.32
35	V--G-2.75	F-1.80	D-1.80
37	S--G-1.28	P-0.71	D-0.60
39	E--G-1.66	K-0.94	D-0.51
38	M--G-3.40	K-2.23	A-1.45
42	R--S-1.83	D-1.38	K-1.30
46	N--G-2.23	D-1.38	H-1.30
52	I--G-3.63	T-3.61	S-2.93
53	D--P-1.12	G-0.62	A-0.41
61	Q--P-1.33	S-1.24	N-1.12
68	T--G-2.22	P-1.11	S-0.83
69	M--G-1.79	S-0.83	K-0.53
72	S--G-1.32	C-0.60	P-0.14
73	Q--N-1.33	G-1.1	S-0.66
74	Y--G-3.83	A-2.13	S-1.57
76	S--G-0.97	P-0.09
77	T--G-2.08	P-1.11	Q-0.52
78	P--G-1.51	A-1.22	D-1.22
79	E--G-1.46	K-1.29	A-0.86
82	A--G-1.29	S-1.09	T-1.08
94	M--G-2.61	S-2.16	K-1.74
107	M--G-1.75	T-1.50	A-1.37
115	L--T-4.84	D-4.39	G-4.17
116	A--K-1.88	H-1.82	T-1.82
120	S--C-1.66	T-1.56	G-1.44
145	Q--N-1.25	G-1.19	P-1.18
147	A--T-1.44	P-1.32	G-1.27
148	V--G-2.62	F-1.72	P-1.66
152	A--T-1.55	P-1.40	H-1.16
154	I--T-3.69	G-3.58	D-3.14

Finally we got the mutated protein sequence whose corresponding protein will have higher thermal stability and the sequence is follows:

MKTPDTEAGP PADPQGRFLSGTELQPA G GRGDSAGA GGGAASALTGNAQSL GPGAAQAVYP KFPYTTGGQGGNG AGGGGGKG KCARDIGYYLRGVTYCLVAGGTGPG DEYLIAGTKEIN SCTFDLSPSWYVEALKYIKANHGLSG N ATGEANTYT DYAINALS

Through site-directed mutation we can predict the sequence of a protein that has the higher stability and the corresponding steps are all similar as we talked above.

Innovation methods in Modeling

Using genome-scale metabolic model, you can predict the quantity of your final product and the influence of your modified pathway to the host’s metabolic flux.

Related study has made great progress in yeast recent years, for example, the paper, [A consensus S. cerevisiae metabolic model Yeast8 and its ecosystem for comprehensively probing cellular metabolism], constructed a model which forms the basis of the model ecosystem. And based on Yeast 8.5, we carried out our innovation work.

Firstly, as presented in the experiment part, new genes are introduced in S288c, so we add the involved reactions and important metabolites to the original network.we use MATLAB cobra toolbox to construct the new network.

Secondly, according to the previous test results, we find the flux of precursor heme b is subtle, so we changed the the constraints of heme b, and use MATLAB cobra toolbox to calculate the flux.

Then, drawing upon the visualization tool fluxer, we mapped the new metabolic networks, and present the flux in the key paths.

The results obtained from models above can guide the experiment, the fermentation model and the practical production.

Construction of 3D-hardware

The constructing of the 3D hardware can guide us to construct our idea of how to implement our project really and making the project possessing integrity.

Firstly, before the building of the factory we should determine the process of our fermentation and draw the fermentation process down. You can refer to the literature reviews and the books as‘Principles of Chemical Engineering’ and so on. Combining the process we learned and the characteristic of our yeast, we finally determined our Phycocyanin-Beer factory as follows.

Now we can set about building our fermentation factory using CAD, 3D MAX or other 3D-modeling software. We learn it by ourselves and through the guidance of professional person we finally construct our factory as follows.

Figure 1. Dynamic representation of our mini factory

Supplement of existing parts

CpcE(CpcT) encodes phycocyanin alpha Phycocyanobilin lyase which is one part of the Phycocyanobilin lyase catalyzing alpha phycocyanin and 3Z-phycocyanobilin. Phycocyanobilin lyase is required for the chromophorylation of α-phycocyanin to form holo-α-subunit. We further improve the expression of the lyase through optimize it in the system of yeast.

Optimized Sequence(Optimized Sequence Length:663bp,GC%:45.25):

ATGTTGTCATTAGAACAATTGGCTACCCAATTGGAATCTCCAAACTCTAGAGACAGATTGTTGGCCCTTGCTGCCTTAAGAGATGTTCCACCTCCAGAAGCTGTTCCATTGATCAAGAA

GGTTTTGTGGGATGACAATTTGCAAATCCGTTCCATGGCTGTCTTTGCTTTGGGTGTTAAGCCTACCCCAGAATGTTTCGACATTTTAGTCCAATTGTTGGAAACTGAACCAGACTACG

GTATCAGAGCTGATGCTGCTGGTGCCTTGGGTTATTTGCAAGACCAAAGAGCCTTCGCTCCATTAGTCAGAGCTTTCTACGAAGACACTGATTGGTTGGTTAGATTCTCTGCCGCTGT

CCATTGTTGAAGAAATCTTGCCATTCGTCACTGCTGAAGACTGGTTGATTAGACAAAGATTAGCCGAAGCCCTAGGTAACTTGCCATCTCCAAAGACCCAATCCGCACTAGAATACTT

GGCTAAGGATGAACACCCACACGTTGCTGCCGCTGCTGAAATCTCTTTGCAACGTTTGGCTCAAAGATGA

If you also want to express the cpcE gene in the host of yeast, the sequence above has been optimized and was already proved to be function.

Team:ECUST China/Contribution

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