iGEM 2020 — Team NJTech_China

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# Team:NJTech China/Notebook

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Notebook

Protocols

Affinity chromatography

Agarose gel electrophoresis

BCA protein assay

Cell Lysis

IPTG Protein Expression Induction

PCR

Product collection and purification

SDS-PAGE verification

Transform BL21

## Agarose Gel Electrophoresis

1% agarose is added to 1 × TBE and heated in microwave for about 1 min to fully
dissolve the agarose. Let it cool for a while, add a small amount of ExGreen
nucleic acid dyeμL, mix them thoroughly and pour the contents into the rubber
plate. The gel is verified and a single band appeared, indicating that the
plasmid is linearized.4μL per well, 1μL 10× Loading Buffer per well.

**Electrophoresis** : 125V 30min

## BCA protein assay

Beyotime BCA Protein Assay Kit

## Cell Lysis

1) Divide the bacterial solution into 50mL centrifuge tubes. Centrifuge at 6000 rpm at 20°C for 10 minutes and pour the supernatant into a waste bucket.

2) Resuspend the cell pellet in 10 mL 10 mM Tris-HCl (pH 7.5), centrifuge at 6000 rpm at 20°C for 10 minutes , and pour out the supernatant. Repeat the above steps again.

3) After centrifugation, the cell precipitation was resuspended in 9.9 mL 10 mM Tris-Hcl (pH 7.5) and 0.1 mL lysozyme , and incubated at 30℃ for 20 minutes.

4) 200 μl 100 mM PMSF was added to the fermentation broth. Remove 200 μl liquid into 2ml centrifuge tube and mark it.

5) Immerse the bacterial suspension in ice and perform sonic treatment with 3 s sonic wave and 3 s interval for a total of 30 minutes.

6) Remove 200 μl of whole protein solution from 50mL centrifuge tube into 1.5mL centrifuge tube and mark it.

7) Centrifuge at 4℃ and 6000 RPM for 20 minutes, take 200 μl supernatant from the 50mL centrifuge tube into the 2 mL centrifuge tube and mark it. Pour the remaining supernatant into the new 10 mL centrifuge tube and wait for ultrafiltration. The precipitate was resuspended with 300 μl pure water in a 2mL centrifuge tube and marked.

## IPTG Protein Expression Induction

1) Transform expression plasmid into BL21. Plate on antibiotic selection plates and incubate overnight at 37°C.

2) Resuspend a single colony in liquid culture with antibiotic to produce a starter culture. Inoculate starter culture into 50 ml expression media containing antibiotic.

3) Incubate at 37°C with shaking at 220 rpm until OD600 reaches 0.6 .

4) For most vector systems, induce with 250 μM IPTG and express protein for 24 hours at 16°C with shaking at 220 rpm.

## PCR

50μL system (2 × Rapid Taq Master Mix):

2 × Rapid Taq Master Mix       25.0 μl

Primer1 (10 μM)                       2.0 μl

Primer2 (10 μM)                       2.0 μl

Template DNA\*                        x μl

Add ddH2O to 20μL

\*For a 50 μL reaction system, the recommended input amount of template is as follows:

|  |  |
| --- | --- |
| Template type | Template start amount |
| Genomic DNA of animals and plants | 0.1-1 μg |
| Genomic DNA of E. coli | 10-100 ng |
| cDNA | 1-5 μl (< 1/10 of the reaction volume) |
| Plasmid DN | 0.1-10 ng |
| λDNA | 0.5-10 ng |

Program:

|  |  |  |  |
| --- | --- | --- | --- |
| Loop step | Temperature | Time | Number of cycles |
| Pre-denatured | 95℃ | 3min | 30-35cycles |
| Changing nature | 95℃ | 30sec |
| Annealing | 53℃ | 30sec |
| extend | 72℃ | 15sec |
| Extend comletely | 72℃ | 5min |

## Product Collection And Purification

### Ultrafiltration：

1) Take appropriate amount of fermentation solution, disperse them into two 50mL centrifuge tubes, centrifuge at 3500 rpm, 10°C for 10 min after equilibration.

2) After centrifugation, the supernatant is poured out, and 50 μL of the supernatant is taken and marked as sample 1;

Take a certain amount of ultrapure water, resuspend the bacteria , and marked it as sample 2;

### The remaining supernatant is subjected to ultrafiltration (the membrane is placed in a 35 mL centrifuge tube):

(1)Add 10 mL of ultrapure water to each of the two ultrafiltration tubes, balance, 3500 rpm, and centrifuge for 20 min;

(2)Add 10 mL of sample 1 to each of the two ultrafiltration tubes, balance, 3500 rpm, centrifuge for 20 min, aspirate the liquid on the filter, and transfer to a 1.5 mL centrifuge tube;

Repeat the operation of step 2 until all the samples 1 are processed, the liquid in the 1.5 mL centrifuge tube is marked as sample 3, and the liquid in the 35 mL centrifuge tube (i.e. the supernatant after ultrafiltration) is marked as sample 4;

(3) Add 10 mL of ultrapure water to each of the two ultrafiltration tubes, level, 3500 rpm, centrifuge for 20 min (clean filter), and immerse the filter in ethanol.

## SDS-PAGE verification

Beyotime SDS-PAGE Gel Preparation Kit ; Gels were washed in pure water and then stained with 0.25% Coomassie Brilliant Blue to reveal protein bands.

## Transform BL21

1) Take the competent BL21 from the -80 °C refrigerator and thaw it on ice.

2) Add the 1-2 μL DNA fragments to be transformed into 50 μL competent cells, mix the
walls of the tube (do not shake and mix), and incubate them on ice for 30
min.

3) Heat-shocking for 90 sec in a 42°C water bath, then immediately set it on ice for 2 min. Do not shake the tube.

4) Add 800 μL LB medium (without antibiotics), shaking at 37°C, 200 rpm for 1 h.

5) 6000rpm, centrifuge for 2 min, discard 800μL of supernatant, suspend the bacteria body with the remaining medium, and evenly coat on the LB solid plate containing the corresponding antibiotics。

6) Incubate the plate in a 37 °C incubator for 10 min. After the bacteria solution is completely absorbed, invert the plate and incubate it overnight.

3.Plasmid Extraction：ZOMANBIO Fast Plasmid Miniprep Kit

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# Team:NJTech China/Engineering

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**Engineering Success**

In short, the purpose of our project this year is to explore the expression ability of natural pheromone-responsive promoters and expand the application range of such promoters through signaling pathway modification. We have designed and characterized artificial promoters according to the role of PREs. Pheromone-inducible promoters show diverse expression capabilities. Promoters with different characteristics can be used for the construction of biosensors and metabolic genetic engineering regulation, and the achievement of intercellular behavior. However, the expensive pheromone limits the application of such promoters in these scenarios. To broaden the application range of pheromone-responsive promoters, we reconstructed the pheromone signaling pathway, trying to build a chassis that responds to cheaper inducers. By reading literature, we learned that Ste5ΔN-CTM protein can activate the MAPK pathway in the absence of pheromone and promote the expression of pheromone-responsive genes. We constructed the Ste5ΔN-CTM strain to enable galactose to induce the expression of mating genes. As a result, the pheromone-responsive promoters can be used for the construction of cell factories.

**Research on PREs**

Promoter engineering provides sophisticated and diverse expression strategies for metabolic engineering and biosensors. Previous studies have demonstrated the valuable application of promoter engineering in the precise control of gene expression levels1. And the result of promoter modification is relatively stable and not easy to lose during cell culture. During the process of yeast mating, there are a series of promoters induced by pheromone. The strength of pheromone responsive promoters is closely related to the arrangement of pheromone response elements (PREs) in these promoters2.

PREs recruit the transcription factor Ste12 which acts as the major transcriptional activator to activate the promoters in mating genes. Pheromone responsive promoters generally contain multiple copies of PREs. PREs can endow the minimal gal1 core promoter with the ability to respond to pheromone. The sequence, copy number, and orientation of PREs may affect the basal and pheromone responsive expression level of promoters3.

Research has shown that the activity of the promoters strengthens as the number of PREs sites increases, but the association between PRE orientation and promoter strength is not clear. This year our project intends to explore the expression ability of natural pheromone responsive promoters and study the impact of the number and orientation of PREs on it. After reviewing the literature, we selected three promoters, which are p*fig1*, p*prm1*, and p*fus2*, as the research objects. These three promoters contained putative PREs with copy numbers of 5, 3, and 2, respectively4-6. In addition to the difference in the copy number of PREs, they also differ greatly in the orientation and sequence of PREs.

![](https://static.igem.org/mediawiki/2020/7/7e/T--NJTech_China--Engineering1.png)

**Fig. 1 PREs in p*fus2*, p*prm1* and pfig1. (A) Two PREs in p*fus2*. (B) Three PREs in p*prm1*. (C) Five PREs in p*fig1*.**

**1.1 Pre-Experiments**

First, the growth characteristic of *Saccharomyces cerevisiae* BY4741 treated by α-factor was evaluated. We conducted halo assay and growth curve measurement. The results showed that α-factor can inhibit the growth of BY4741 on solid and liquid YPD medium. The effect of pheromone in inhibiting growth is positively related to its concentration. These experiments laid an experimental foundation for our later promoter characterization experiments. We chose the concentration with an obvious difference in growth inhibitory effect to set up the subsequent comparative experiments.

![](https://static.igem.org/mediawiki/2020/3/31/T--NJTech_China--Engineering2.png)

**Fig. 2 Halo assay of yeast BY4741**

![](https://static.igem.org/mediawiki/2020/d/d4/T--NJTech_China--Engineering3.png)

**Fig. 3 The growth curve of yeast BY4741 treated by different concentrations of pheromone**

**1.2 Characterization of three natural pheromone responsive promoters**

Next, we used GFP reporter genes to characterize pheromone responsive promoters, and constructed engineering yeast strains containing GFP reporter regulated by p*prm1*, p*fig1*, or p*fus2*. After pheromone induction, the intensity of these promoters is reflected by the fluorescence intensity of GFP which is quantified by flow cytometry. Then, we analyzed the intensity of promoters at different pheromone concentrations, demonstrating the diverse expression capability of pheromone responsive promoters. When OD=0.6, α-factor was added for induction. Results showed that the pheromone has a significant inducing effect on these three promoters. Within a certain range, which is among 1-5 ![](https://static.igem.org/mediawiki/2020/9/97/T--NJTech_China--EngineeringIcon1.png), the higher the concentration of pheromone, the more obvious the induction effect is. Comparing the GFP expression level of these three promoters, p*fus2* is the strongest, followed by p*fig1*, and p*prm1* is the weakest.

![](https://static.igem.org/mediawiki/2020/5/53/T--NJTech_China--Engineering4.png)

**Fig. 4 The fluorescence intensity of GFP expressed by different promoters induced by different concentrations of pheromone**

**1.3 New findings from promoter characterization experiments**

We summarized several conclusions that have not been reported before. According to previous predictions, the expression level of p*fus2* should be the weakest, but the experimental results showed that the intensity of p*fus2* was higher than p*prm1*. According to the literature and database, the two PRE sites of p*fus2* have opposite directions, while the three PRE sites of p*prm1* are in the same direction. It has been reported that the expression level of the promoter with the PRE sites in the "tail-to-tail" orientation is higher than that of the promoter with the PRE sites in the "same direction" orientation, and our experimental results are consistent with this. The specific influence caused by different PRE orientations needs to be investigated and verified by further experiments.

**1.4 Modification of natural promoters**

Based on the characterization results, we found that although p*prm1* possesses three PREs, it has a low induced expression level which indicates a good potential for modification. The background expression level of this promoter is low which means it can be used to express some proteins that require lower concentrations in the early stage. Based on the literature, we learned the general effect of PREs
on promoter strength. For example, an increase in the copy number of PREs can enhance the induced expression level of the promoter. And a PRE which is aligned in the direction of the promoter may be more effective than a PRE which is in the opposite direction of the promoter3. In the first modification scheme, we doubled the 3 × PRE sequence in natural p*prm1*. The modified p*prm1* is referred to as p*prm1* Ultra which contains 6 × PREs. In the second modification scheme, we changed the orientation of the 3 × PREs in natural p*prm1*. The modified promoter is referred to as p*prm1* Pro which contains three PREs oriented in the same direction as the promoter. As a consequence, the two modified promoters exhibit characteristics remarkably different from the wild type. Unexpectedly, the activity of p*prm1* is higher than that of the p*prm1* Pro and that of the p*prm1* Ultra under the treatment of high concentration pheromones. The activity of the p*prm1* Ultra is higher than that of the p*prm1* Pro, which means that the orientation of the PRE site on the promotor is critical to the expression level of the promoter. Although the experimental results did not meet our expectations, this result still provided some interesting conclusions. e.g.（Please see the Gold Award section for details：<http://parts.igem.org/wiki/index.php?title=Part:BBa_K3384314>）

![](https://static.igem.org/mediawiki/2020/b/b4/T--NJTech_China--Engineering5.png)

**Fig. 5 Improvement of p*prm1***

![](https://static.igem.org/mediawiki/2020/f/f2/T--NJTech_China--Engineering6.png)

**Fig. 6 The fluorescence intensity of GFP expressed by different modified p*prm1* induced by different concentrations of pheromone**

Taking the experimental period and other factors into account, we only selected a few pheromone concentrations gradient for the experiment. At the same time, our experimental sample size is limited.

**1.5 The application of Mathematical models in our project**

Mathematical models are another important tool for characterizing promoters. In our project, neural network algorithms can predict the expression level of pheromone-inducible promoters. The molecular dynamics model guides the design of experimental conditions and is the basis for obtaining a relatively accurate pheromone concentration profile of the promoter through as few experiments as possible.（ [https://2020.igem.org/Team:NJTech\_China/Model](https://2020.igem.org/Team%3ANJTech_China/Model) ）

**Construction of the Ste5ΔN-CTM strain**

**2.1 Inspiration and design**

![](https://static.igem.org/mediawiki/2020/c/c9/T--NJTech_China--Engineering7.png)

In the course of the experiment, we found that the price of pheromone is much higher than that of the galactose. The price of 10mg of the pheromone can buy nearly 800g of the galactose. We hope to modify the yeast pheromone signaling pathway so that other inducers can activate the expression of pheromone-responsive promoters, thereby cutting the expenses in some biological experiments. The pheromone signaling pathway is a process of coupling between G protein and MAPK pathway. Among them, Ste5 plays an important role responsible for associating the G protein with the MAPK component in the signaling pathway. It can be seen that the modular structure of Ste5 provides possibility for the modification of the signaling pathway. Inspired by this, we intended to construct an engineering  *S. cerevisiae* strain as a chassis cell for research, in which the pheromone responsive promoters can be induced by the cheap inducer galactose. After reviewing the literature, we found a Ste5 mutant called the Ste5ΔN-CTM7. The 214 amino acids at the N-terminus of Ste5 contain membrane binding sites and mediate the membrane localization of Ste5 under the control of Gβγ. By deleting the N-terminal sequence, the activation of Ste5 is independent of G protein. The C-terminal transmembrane domain of Snc2 was added at the Ste5ΔN C-terminus in order to constitutively recruit Ste5 to the plasma membrane. Ste5ΔN-CTM protein can activate the MAPK pathway in the absence of pheromone and promote the expression of pheromone-responsive genes.

**2.2 Construction of Δste5 strains**

We aim to eliminate the interference of natural Ste5 on the modified signaling pathway. Initially, we learned that the CRISPR-Cas9 system is widely applied to gene knockout in *S. cerevisiae*, so we decided to try the CRISPR-Cas9-based gene knockout technology. Firstly, we used ChopChop and Benchling to design gRNA, constructed recombinant plasmids containing gRNA and carried out yeast transformation. But the PCR results showed that the gene knockout was unsuccessful. We sequenced the pCas9 plasmid and found some problems with its sequence. We will analyze the specific reason for failure and modify the plasmid.

To achieve the *Δste5* strains, we used homologous recombination for gene knockout. Through nucleic acid electrophoresis and sequencing, we filtered out the *Δste5* strain. We named it as *ste5*Δ-loxp-1.

![](https://static.igem.org/mediawiki/2020/2/24/T--NJTech_China--Engineering8.png)

**Fig. 7 Nucleic acid electrophoresis verification of strain *ste5*Δ-loxp-1**

To further verify the knockout of *ste5*, we conducted a Halo assay experiment. According to the related literature, if the haploid yeast lacks the Ste5 protein, the yeast will not be able to respond to the pheromone, and the cell growth will not be inhibited, failing to form a halo on the plate. The *ste5*Δ-loxp-1 strain did not form a halo on the plate. This result is in line with the description of the *ste5*-deficient yeast strain in the literature.

![](https://static.igem.org/mediawiki/2020/7/7f/T--NJTech_China--Engineering9.png)

**Fig. 8 Halo assay of strain *ste5*Δ-loxp-1**

**2.3 The construction of pheromone-independent Ste5 highly active mutant**

After the construction of the Δ*ste5* strain, we introduced the p*gal*1-Ste5ΔN-CTM gene into it, and named it the pheromone-independent Ste5 highly active mutant, PIDS for short. The engineered yeast can transform from the vegetative phase to the mating phase under the induction of galactose. To further characterize the induction of PIDS by galactose, we plan to transform the pheromone responsive GFP reporter gene into PIDS to quantitatively evaluate the signal transduction level of this mutant. We spread the PIDS strain on a plate with galactose as the sole carbon source.

According to relevant literature, the strain should not be able to grow on the plate. Our experimental results are consistent with the theory. The results of the experiment are shown in Figure 9. The left side of the figure is the PIDS strain and the right side is the BY4741 strain. It can be clearly seen that there is no colony growth on the left side and the yeasts on the right side grow well.

![](https://static.igem.org/mediawiki/2020/b/b9/T--NJTech_China--Engineering10.png)

**Fig. 9 The growth of the Ste5 ΔN-CTM strain and BY4741wild type strain on the plate with galactosyl as the sole carbon source**

![](https://static.igem.org/mediawiki/2020/a/a2/T--NJTech_China--Engineering11.png)

**Fig. 10 The growth of the Ste5ΔN-CTM strain and BY4741 wild-type strain
on the plate with glucose as the sole carbon source**

As shown in figure 9, the left side of the plate is the PIDS strain and the right side is the BY4741 strain. It can be clearly seen that there is no colony growth on the left side and the yeast on the right side grows well. Meanwhile, as shown in figure 10, on SC complete medium, both PIDS strain and BY4741 strain grew well.

**References**

[1] Xu, N., Wei, L., and Liu, J. (2019) Recent advances in the applications of promoter engineering for the optimization of metabolite biosynthesis, *World J Microbiol Biotechnol 35*, 33.

[2] Sengupta, P., and Cochran, B. H. (1990) The PRE and PQ box are functionally distinct yeast pheromone response elements, *Mol Cell Biol 10*, 6809-6812.

[3] Su, T. C., Tamarkina, E., and Sadowski, I. (2010) Organizational constraints on Ste12 cis-elements for a pheromone response in *Saccharomyces cerevisiae*, *FEBS J 277*, 3235-3248.

[4] Elion, E. A., Trueheart, J., and Fink, G. R. (1995) Fus2 Localizes near the Site of Cell-Fusion and Is Required for Both Cell-Fusion and Nuclear Alignment during Zygote Formation, *Journal of Cell Biology 130*, 1283-1296.

[5] Erdman, S., Lin, L., Malczynski, M., and Snyder, M. (1998) Pheromone-regulated genes required for yeast mating differentiation, *J Cell Biol 140*, 461-483.

[6] Heiman, M. G., and Walter, P. (2000) Prm1p, a pheromone-regulated multispanning membrane protein, facilitates plasma membrane fusion during yeast mating, *J Cell Biol 151*, 719-730.

[7] Lamson, R. E., Takahashi, S., Winters, M. J., and Pryciak, P. M. (2006) Dual role for membrane localization in yeast MAP kinase cascade activation and its contribution to signaling fidelity, *Curr Biol 16*, 618-623.

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# Team:NJTech China/Experiments

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**Experiments**

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# Team:NJTech China/Results

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**Results**

**1. Characterization of pfus2, pprm1 and pfig1**

**2. Construction and Characterization of pprm1 Ultra and pprm1 Pro**

**3. ste5 Knockout**

**4. Construction and Characterization of PIDS**

**5. amajLime Characterization**

**6. eforRed Characterization**