Difference between revisions of "Team:Tsinghua-A/Achievements"

Revision as of 00:34, 18 October 2018

Modification of the yeast

Gene knockout

From the researches before, in order to reduce the loss of the signal strength, three gene are recommended to be knocked out.

Ste2, the receptor of α factor, in order to eliminate the probability of yeasts getting affected by native ligand-pheromone.

Sst2, the GTPase-activating protein for Gpa1p, as a receptor-desensitizing factor in order to eliminate possible up-stream GPCR signal conductive decline due to incessant stimulation.

Far1, the cyclin-dependent protein serine/threonine kinase inhibitor, in order to eliminate side effect to cells’ life circle and formation due to signal intensification in MAPK pathway of the pheromone pathway.

Fortunately, we found that BIT-China 2017 used the same pheromone pathway in yeast in their project last year, so we established cooperation with them and brought their yeast-- CENPK2-1C ura3::far1 his2::sst2 trp3::ste2. It greatly reduced our experiment works.

Mutation screening

With CENPK2-1C ura3::far1 his2::sst2 trp3::ste2 △leu2 Saccharomyces cerevisiae, we thought the auxotroph marker is not enough for subsequent experiment, on the other side resistance markers have a few problems in yeast, such as the plasmids are lost easily. So we used positive 5-fluoro-orotic acid (5-FOA) selection to obtain Ura3 mutant strains. In this method, Wild-type strains of yeast (or ura3 mutant strains containing a plasmid-borne URA3+ gene) are unable to grow on medium containing the pyrimidine analog 5-fluoro-orotic acid, whereas ura3- mutants grow normally [1].

CENPK2-1C ura3::far1 his2::sst2 trp3::ste2 △leu Saccharomyces cerevisiae were plating on the ura+, his-, trp-, leu+ solid medium containing 5-FOA, monoclonal was obtained and enlarge cultivated. Then plated the yeast on ura-, his-, trp-, leu+ solid medium, no colony can be observed to survive on it. For further confirm, the colony’s genome was sent for sequencing, mutation existed in Ura3 gene. We considered that we had gained CENPK2-1C ura3::far1 his2::sst2 trp3::ste2 △leu2 △ura3 Saccharomyces cerevisiae.

                           <img src=" 

Plasmid construction

In our project, these plasmids are required.

Receptor plasmid

Coding sequencing of Human histamine receptor (HRH4) was got from plasmid pH4R-mCherry-N1. Separately, HRH4 CDS and HRH4-mCherry fusion sequencing were obtained by PCR. We chose pGADT7-AD (Leu2 marker) and use HindIII to remove SV40-NLS sequencing, GAL4 activation domain and HA tag. Then insert HRH4 or HRH4-mCherry between ADH1 promoter and ADH1 terminator.

                           <img src=" 

pGADT7-HRH4-mCherry was used to express HRH4-mCherry fusion protein to show the expression and localization of HRH4 in yeast. pGADT7-HRH4 was used to express functional HRH4.

Reporter

We used part: BBa_K775004 as our reporter, because it was not released, so we used PCR to get Fus1 promoter from yeast genome, EGFP and ADH1 terminator from other plasmids, then used Gibson assembly it with EGFP and inserted into plasmid pYSE2.0 digested with SpeI and EcoRI (Ura3 marker).

                           <img src=" 

Transformation of the yeast

pGADT7-HRH4-mcherry was transformed into CENPK2-1C ura3::far1 his2::sst2 trp3::ste2 △leu2 △ura3 Saccharomyces cerevisiae to test the expression and localization of HRH4-mcherry.

pYES2.0-pfua1-EGFP was transformed into original CENPK2-1C △his2 △trp3 △leu2 △ura3 Saccharomyces cerevisiae to test the reporter.

Both pGADT7-HRH4 and pYES2.0-pfua1-EGFP were transformed into CENPK2-1C ura3::far1 his2::sst2 trp3::ste2 △leu2 △ura3 Saccharomyces cerevisiae to consist our complete yeast for histamine detection. For confirming both of these two plasmids were transformed successfully, colony PCR was performing to detect the existence of these two plasmids.

                           <img src="  " alt=""height="700" width="700"/>

                           <img src=" 

Test and Result

To insure our system can work as we expected, we did a serious of test.

HRH4-mCherry expression and localization

After transforming the CENPK2-1C ura3::far1 his2::sst2 trp3::ste2 △ura3 Saccharomyces cerevisiae with plasmid pGADT7 contains part BBa_K2583001, 30℃ cultivate on △leu solid medium for one day. Select monoclonal and enlarge culture for several days. We chose to feature the expression of H4 at a relatively stable stage to make the result more referable for the modeling analysis.

Location observation by fluorescence microscope

We observed the fluorescence under the microscope with 580nm exciting light. The red fluorescence shows the position of HRH4-mCherry fusion protein.

                           <img src=" 

The location of HRH4 indicates that our pioneering work to try to produce Histamine receptor H4 from Homo sapiens in Saccharomyces cerevisiae was basically a success. Because the function of HRH4 requires at least that the protein should be sent to the membrane.

                           <img src=" 

Quantification by Flow CytoMetry (FCM)

From the result of fluorescence microscopy, the expression of HRH4-mCherry fusion proteins can be observed roughly, for quantifying the expression level of HRH4-mCherry, FCM is performed. From the result, a distinct peak value can be observed. Considering the fluorescence microscopy result that most of HRH4 are correctly located, this data does provide us a reliable probability distribution of total H4 protein expression variance between yeast individuals. Finally, the probability distribution of functional H4 receptor expression variance can refer to the total expression probability distribution we get from experiments.

                           <img src=" 

The test of this composite part set the foundation of our project, it proves that HRH4 can be expressed in high level under the trigger of a constitutive promoter-ADH1 promoter. Besides, the HRH4 can locate in the yeasts’ membrane without adding other sequence. It can be part of the evidence that our HRH4 is functional. Then the Flow CytoMetry result provide us a reliable probability distribution of total H4 protein expression variance between yeast individuals, which is significant for modeling.

The induce of the reporter

To test the ability of responding to upstream pathway, we try to provide valid upstream signal to reporter gene. So we transform the CENPK2-1C △ura3 △leu △his △trp Saccharomyces cerevisiae with plasmid pYES2.0 contains part BBa_ K775004, 30℃ cultivate on SD-ura solid medium for one day. Select monoclonal and enlarge culture for several days. And then, several experimental groups are set to get CENPK2-1C stimulated by α factors. 1ml of modified yeast is centrifuged at 5000 g for 2 min as well as 2 ml of MATα yeast, then supernatant of modified yeast is discarded and 2 ml of MATα yeast supernatant is added to culture yeast.

EGFP expression, Chemotropic Growth and Mating Differentiation observation by fluorescence microscope every hour since the addition of MATα yeast supernatant.

                           <img src=" 

Compared with the Non-Induction group, the EGFP intensity inside the yeast gets more and more stronger from the 1h group to 5h group. And, more and more yeast cells in sight finish the process of mating: stretching out mating projection or can say “shmoo,” and then contacting and fusing between mating partners.

But when time goes to 6h, we can see that, basically the EGFP protein particles in sight are all distributed outside the yeast cell. We suppose that those EGFP protein particles are excreted by yeast cells for preparing to enter ascospore period.

Time scale of EGFP expression upon induction of α factor

Similar to the experiment above, we set different culture group and time to measure the time scale of EGFP expression. The sample order is as follow:

                           <img src=" 

                           <img src=" 

                           <img src=" 

As can be seen from Fig.5 and Fig.6, we can see clear distribution between the induction group and non-induction group. As for the expression time scale, the supernatant induction group reached the peak at 6h while the MATα yeast induction group maintain at a rather high level starting from 3h.

The above result indicate that the reporter plasmid could function as expected by responding to upstream signal in pheromone pathway, thus could be used to modify yeast to target histamine.

The induce of modified yeast with histamine

After successfully transferred the plasmid ADH1-HRH4-Ter and pFUS1-EGFP into CENPK2-1C ura3::far1 his2::sst2 trp3::ste2 △ura3 Saccharomyces cerevisiae, we had already finished the modification process of yeast. Now it’s time to test whether our diagnostic yeast works! Based on our previous design, the modified yeast could respond to different ratio of histamine in environment at corresponding EGFP expression level. At meantime, the protein expression rate is also influence by culturing time after first induction. Due to the modelling, the higher the histamine concentration is, the faster yeast will respond and so is the protein degradation rate. So we made a prediction that the EGFP expression rate would positively correspond to histamine concentration in a certain time period after histamine induction.

Based on the above assumptions, we chose to induce the modified yeast already cultured to logarithm phase in medium lacking His, Trp, Leu and Ura. Then a histamine concentration gradient was made by adding a diverse range of histamine solution to the culture medium. According to medical research, the histamine concentration in normal human blood is around 30 ng/ml to 60ng/ml, while in allergic people, this concentration would reach 100 ng/ml or even more. We set three group of histamine concentration: 0 ng/ml as control group, 50 ng/ml to simulate the normal concentration and 200 ng/ml to simulate allergic concentration.

According to the test of reporter plasmid, we estimated the expression peak would be between 5 to 10 hours, so we choose 5h, 8h and 11h as three time points to run flow cytometry.

One thing worth mentioning is that due to the low concentration of yeast, we could only set 10 group so as to meet the criterion concentration of FCS.

The group set is as follow:

                           <img src=" 

The concentration of yeast is around 100000 cell/ml (measured by OD600). We pick 10000 events inside the gate for each sample during FCS.

The FCS result is as follow:

                           <img src=" 

From Fig.8, we can see that at 5 to 8 hours’ culturing, the EGFP expression rate is positively correspond to histamine which certify our theory. When we judge each concentration group separately, it can be assumed that 50 ng/ml and 200 ng/ml group both reach its peak during 5 to 11 hours’ period. The degradation rate of each group is also positive correspond with histamine concentration, since at 11h, the expression level of every induction group has fallen, and the expression rate appears to be rather negative correlated to histamine concentration.

Considering the fact that the reaction time we had estimated for our allergy test kit is around 4 to 5 hours, it turned out that the modified yeast could indeed distinguish between allergic state blood serum and normal blood serum.

Growth curve of yeast in blood serum

In order to certify the feasibility of yeast maintaining its previous cell activity while reacting with histamine in blood serum, rather than become incompetent or being precipitated by antigens such as IgG in the environment, we carried out a series of experiment by culturing modified yeast in different ratio of blood serum. The ability of proliferation as well as cell activity are revealed by determination of growth curve in different groups.

One thing worth mentioning is that the blood serum (BS) we use is derived from mice with no immunogenicity. After extracted from mice, the product simply went through the procedure of precipitation and bottled without heat inactivation. Control group was set by filter sterilized half of the blood serum (FBS) to remove large protein and potential bacteria (. This procedure aims to judge whether the complement protein would influence the growth of yeast.

The experiment is designed as follows.

                           <img src=" 

Due to the fact that normally yeast would enter logarithmic growth phase in 4 to 8 hours, we choose 1~7 h as the 1st stage measure point and 18~19h as the 2nd stage measure point to judge whether the yeast has entered plateau phase. The density of yeast is measured by OD600.

                           <img src=" 

As can be seen from Fig.9, the addition of blood serum didn’t influence the growth of yeast significantly.

                           <img src=" 

On the contrast, the addition of blood serum did enhance the proliferation of yeast when comparing the curve of H and G with C and D. What’s more, comparing the curve of adding the original blood serum or sterilized one, we can see that the original blood help yeast to grow better without any precipitation.

                           <img src=" 

Compared the curve of original blood serum culturing and the sterilized one, it is obvious that the yeast cultured in original product outgrew the sterilized group in the first few hours. Though the two group reach the same level after 19 hours culturing, we could still judge the original blood serum to be better since the reaction time we had estimated for our ATM testing box is around 4 to 5 hours ---- still within the dominant period of original blood serum culturing.

                           <img src=" 

The overall graphic shows that the growth rate of yeast in YPD is faster than water, while during the estimated reaction time, the whole blood serum group also outgrew the water group. The above results suggest that blood serum won’t disturb the proliferation and cell activity of yeast, and even enhance them in water environment. The dominance of original blood serum leads to convenience that users don’t need to filter or heat inactivate complement protein to avoid precipitation.

In conclusion, the modified yeast could survive in blood serum environment provided by users which is the testing environment in our design normally, without any need of further processing.

Future Work

Gα modification

Because human G protein coupled receptors (GPCRs) can‘t couple with α subunit of yeasts’ G protein. In order to enhance the coupling, the five carboxy-terminal amino acid residues of the guanine nucleotide binding protein a-subunit (Ga) in yeast Gpa1 can be replaced by human Gα[2]. Human histamine H4 receptor is coupled to Gαi/o proteins [3], so we got the sequence of last five amino acid residues of Gi1 and add it to the primer. Then we used PCR and Gibson assembly to constitute fragment for homologous recombination.

Reference

1. Boeke JD, LaCroute F, Fink GR. 1984. A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Molecular & general genetics MGG 197: 345–346.
2. Ishii J, Oda A, Togawa S, et al. Microbial fluorescence sensing for human neurotensin receptor type 1 using Gα-engineered yeast cells[J]. Analytical Biochemistry, 2014, 446:37-43.
3. Thangam EB, Jemima EA, Singh H, et al. The Role of Histamine and Histamine Receptors in Mast Cell-Mediated Allergy and Inflammation: The Hunt for New Therapeutic Targets. Frontiers in Immunology. 2018;9:1873. doi:10.3389/fimmu.2018.01873.
4. Hirashima K, Iwaki T, Takegawa K, Giga-Hama Y, Tohda H. 2006. A simple and effective chromosome modification method for large-scale deletion of genome sequences and identification of essential genes in fission yeast. Nucleic Acids Res 34: e11