Difference between revisions of "Team:BIT-China/Project/"

COOPERATION WITH OTHER UNIVERSITIES

At the latter phases of our project, we organized Beijing University, Lanzhou University and Jilin University etcetera to make a big chat about what we had done so far and held three scientific speeches to introduce the development and the common use of sweeteners in our daily life, which contributed to the increase of popularity of iGEM as well as Synthetic Biology.Ultimately, we collected the critical advice from the audiences in time and accept one of them, which recommended us to set saccharose as the standard, after that running the test would be more reasonable.

SWEETNESS AND WE

Sweetness, a word that is associated with happiness and satisfaction. For most higher animals, it is innate that they prefer the sweetness. Human is not an exception because people are always addicted to the sugar. When we have sugar, our brains will release serotonin, which can make us feel delightful.

WELL WHY?

However, according to the data from the World Health Organization (WHO), having excessive sugar is more harmful than smoking. Having excessive sugar can not only cause obesity, obviously, but also can trigger diseases, such as diabetes and gout.

With the development of technology, artificial sweeteners are becoming better choices. The production technology of artificial sweeteners is becoming more mature. Meanwhile ,as a food additive, artificial sweeteners are also more widely used in the field of food production. But the potential harm of artificial sweeteners to humans is still a controversial issue.

Therefore, it is imperative to detect the ideal sweetener to tackle the problems we mentioned above as well as satisfy our gustation.

THEN WHAT?

With the development of synthetic biology, the types and production of natural products of microbial synthesis are increasing. Does the natural product produced by the microorganism contain some undiscovered sweeteners? Thus we hope to design a system which can identify sweeteners and be able to distinguish the sweetness of these substances by corresponding signal expression.

First and foremost,we must understand the mechanism that people can feel sweetness: the human sweet G protein-coupled receptor T1R2-T1R3. It is a pair of extracellular proteins with homodimer，which has six structural domains. Different sweet substances combine with different regions of T1R2-T1R3，then triggering intracellular G protein coupled pathway and leading to downstream signal expression. Finally, it can lead to a "sweet" feeling.

At the same time, we chose yeast cells as host. We hope to replace its original G protein-coupled receptor Ste2 , which is related to yeast’s mating with the T1R2-T1R3. And some side effects genes are knocked out by homologous recombination technology. After building a complete system, we hope to judge whether the material has sweetness according to whether the signal can express. If there is a response, we hope to judge the sweetness of the substance by the strength of the signal.

BUT HOW?

Our experimental part is divided into three parts:

Host Reconstruction: host reconstruction group uses homologous recombination methods ,using Trp, His, and Ura as screening marker fragments with homologous arms to knock out Ste2, Sst2, and Far1 genes in yeast.Except Ste2 , Sst2 gene can inhibit the signal transduction of Gpa1 protein.Far1 gene avoids interference signal for sweet yeast yeast normal physiological function after transformation.

Receptor Expression: the receptor expression group uses OE, PCR, and other methods to achieve the synthesis of T1R2-T1R3 G protein coupled receptors. The detection team constructed the pFUS and the red fluorescent protein line to detect the sweetness and sweetness functions through the expression of the fluorescent signal.

Detection of Circuit:in combination with our project, our mathematical modeling also establishes three sets of models that provide guidance for our experiments and achieve the desired functional simulation.

Human sweet receptor T1R2-T1R3 heterologous expression

To synthesis the target gene, Human sweet receptor T1R2-T1R3, we searched gene from web site NCBI firstly, then we use the software Snapgene to optimize the sequence. The way we choose to synthesis the target gene is to use oligo design software OLIGO.

First we design 90 primers for each sweet receptor and each primer has 15bp overlap region with the adjacent one. Next we divided 90 primers into 3 groups，named block A、B and C，mix the primer of each group and made the final concentration to 10μm. We successfully get 6 desire fragments after PCR and purification.

To verify whether the heterodimer is successfully expressed and located at the certain position, we add different color protein as tags to each sweet receptor. After checking the part library，we get blue, yellow and green fluorescent tags. During the primer designing, we add another 20bp of N-terminal of each sweet receptor as the overlap region. So through OE-PCR we can link fluorescent tags to each sweet receptor.

Another way we design to verify whether the heterodimer is successfully expressed and located at the certain position is that to add different epitope tags instead the color protein to each sweet receptor. The reason we choose two different methods is that according to the protein translation process， add color protein on the N-terminal of the target tagging protein may not give color protein the enough time to fold as it’s functional state before it been secretion， thus a his tag along with antibody detection maybe a better solution.

In order to express the human receptor, we choose Saccharomyces cerevisiae strain Cen.PK2-1C as our host and the shuttle vector, pESC-Leu, to express T1R2-T1R3. We select two restrict sites for two receptor genes respectively. Then we use PCR to add BamHI and SalI to the T1R2 fragment. In the meantime, we add SpeI and NotI to T1R3 fragment. And the fragments with fluorescent tags or other tags also link to restrict sites through PCR.

After using T4 ligase for ligating the digested DNA fragments to the multi cloning sites of plasmid pESC-Leu, we transform the ligation production to the E.coli TOP 10 then select the positive colony using colony-PCR.

After finishing the construction of vector with sweetness receptor in E.coli, the recombination plasmid is transformed into yeast, Cen.PK2-1C with pFus-RFP-Cyc1t(G418 resistance vector)， through LiAc transformation. Because of the auxotrophic selection marker Ura in the pESC-Ura, the colony is chosen under the SD-Uracil defect medium with 0.3% G418. Then the positive cloning can survive in this medium. In this way, we can get the reconstruction Saccharomyces cerevisiae successfully.

Since the T1R2-T1R3 with fluorescent tags is designed to confirm the expression and location of T1R2-T1R3 in the Cen.PK2-1C, we test our yeast by fluorescence plate reader through the immunofluorescence technology. We use the minimal induction medium to induce the Gal 1/10 promoters and express their downstream gene then detect the fluorescence

Protocol for fluorescence test through fluorescence plate reader (Red fluorescence protein):

1. Incubate the yeast for 24 hours preciously using SD-Uracil defect medium with 0.3% G418 to get the harvest the cells.

2. Replace the SD medium by minimal induction medium:

yeast ni- trogen base without amino acids (YNB)	6.7g/L
Necessary amino acid mixture (without Leu、Trp、Ura、His)	1.3g/L
Galactose	2%
Glycerol	2%
Leucine	0.1g/L
Tryptophan	0.04g/L
Histidine	0.02g/L

3. Culture the yeast for 12 hours(这个可能会变).

4. Sampling and test the fluorescence adsorption value (FAV) by plate reader. The fluorescence excitation wavelength is 4884 nm and the fluorescence adsorption wavelength is 575 nm.(The excitation/absorption wavelength of different fluorescence protein is chosen according to their characteristic excitation/adsorption.Our antibody is PE conjugate goat anti-mouse IgG)

In order to enhance the signal transmission and amplification, we need to knock out some genes of yeast, including Ste2, Sst2 and Far1. Ste2, Sst2 and Far1 exist in endogenous GPCR pathways of yeast, which responds to the GPCR signal and start the downstream of the Signaling pathways. Ste2 interferes with the sweetness signals which respond to the pheromone. Knocking out Sst2 is necessary to remove the inhibition of sweetness signals. Far1 regulars the cell division which may have a bad influence on the yeast after the genes are knocked out. All in all, knocking out these genes is an essential step of the experiment. The yeast can cause the gene replacement or gene lost by homologous recombination, for that reason these genes will be knocked out by the means of designing the primers cleverly.

To make our yeast detect sweeteners, we replace ste2 receptor with Human sweet receptor T1R2-T1R3. So, first, we have to knock out ste2 gene. To decrease other interference factor we knock out sst2 gene and far1 gene. sst2 and far1 are genes that functioned to tunning signals among the MAPK pathway, they are believed to coordinated growth while yeast’s mitosis as described in the Design.

By NCBI databases for obtaining ste2 sst2 and far1 gene sequence information, we find appropriate homologous arm in upstream and downstream genes. In order to knock out these genes effectively, we select different lengths of homologous arms, including 50bp, 200bp and 500bp. According to the wet-lab measurement, we find out 200bp and 500bp homologous arms are more efficient.

We design 3 pairs of primers cleverly whose templates are the upstream homologous arm, marker and the downstream homologous arm respectively. The markers are Histone synthesis gene, Uracil synthesis gene and Trptophan synthesis gene, short termed His, Ura and Trp . His will replace sst2, Ura will respanlace far1 and Trp will replace ste2. Initially, we get 3 fragments by PCR and they have the overlap areas with each other.

Secondly, the complete fragment observed by OE-PCR is converted to the yeast which has been already knocked out the endogenous gene Uracil, Histone and Trptophan. Additionally, the colony is chosen on the relevant nutritional deficiency medium, so that only the positive cloning can survival on it.

To verify whether the gene is actually knocked out and avoid the false positive colonies, we design the primer1,2,3 and 4 for each genes, as shown in the figure1. The primer 1 and primer 4 are on the yeast genome. The primer 2 is on the marker and primer 3 is on the gene which will be knocked out.

If we get correct results by the primer 1 and 2 as well as nothing from primer 1 and 3, demonstrate that the gene is knocked out. Then we sequencing the PCR product using primer 1 and 4 to make sure the sequence is right.click here to read results

After knocking out far1, we test the growth curve of the yeast. Far1 protein is a cyclin-dependent kinase inhibitor. Deletion far1 gene can relieve inhibition. As you can see, it’s……

After knocking out sst2, we test the growth curve of the yeast. Sst2 protein is an important negative regulatory factor of GPSTP. When we knock out sst2 gene, the sensitivity of the yeast GPSTP could be improved. And the inhibition of cell growth will be enhanced.

As you can see, ΔSst2 strain is much more sensitive to α pheromone. Compared with CENPK2-1C strain, less amount of α pheromone can cause yeast growth arrest.

The signal reporter device

To measure the sweetness of sweeteners, we design the signal reporter device. The device consists of a promoter pFUS, a reporter gene mRFP, and a terminator CYC1t.

When the human sweet receptor T1R2-T1R3 detects the sweeteners, the signal can be transmitted to this device through the MAP kinase pathway which exists in yeast naturally. And this pathway activates the promoter pFUS specificitly, thereby initiating the expression of the reporter gene.

In order to construct this device, first, we connect three parts (pFUS, mRFP, CYC1t) together by OE-PCR. But the result of this procedure is always fail. Then, we change to use Gibson assembly to connect this device with the linear plasmid pRS42K. pRS42K is a kind of shuttle vector using between E.coli and yeast. After finishing the construction in E.coli, we transformed the plasmid into competent cell Cen.PK2-1C, the mating a type haploid Saccharomyces cerevisiae.

In order to find out whether the signal reporter works or not, we cultivate two kinds of haploid S.cerevisiae, Cen.PK2-1D(αtype) and Cen.PK2-1C(a type) together and observe them by fluorescence microscopy. The Cen.PK2-1D(a type) can excrete the a pheromone which can be detected by the pheromone receptor Ste2 positioning in the membrane of Cen.PK2-1C(a type). After detecting the signal, our reporter device will express RFP. Besides, we also use purified α pheromone to test our device’s function.

Group A: transformated CENPK2-1C(a type) and CENPK2-1D(a type)

Group B: transformated CENPK2-1C(a type) alone

Group C: CENPK2-1D(a type) alone

After 9 hours, group A was fluorescent, and group B and group C didn’t fluoresce. The result means signal reporter device worked.

In order to construct this device, first, we connect three parts (pFUS, mRFP, CYC1t) together by OE-PCR. But the result of this procedure is always fail. Then, we change to use Gibson assembly to connect this device with the linear plasmid pRS42K. pRS42K is a kind of shuttle vector using between E.coli and yeast. After finishing the construction in E.coli, we transformed the plasmid into competent cell Cen.PK2-1C, the mating a type haploid Saccharomyces cerevisiae.

In order to find out whether the signal reporter works or not, we cultivate two kinds of haploid S.cerevisiae, Cen.PK2-1D(αtype) and Cen.PK2-1C(a type) together and observe them by fluorescence microscopy. The Cen.PK2-1D(a type) can excrete the a pheromone which can be detected by the pheromone receptor Ste2 positioning in the membrane of Cen.PK2-1C(a type). After detecting the signal, our reporter device will express RFP. Besides, we also use purified α pheromone to test our device’s function.