Team:Jiangnan China/Engineering

Jiangnan_China

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  Integrated Human Practices Education & Science Communication Collaborations Guidance For Cosmetics

• Home
• Project
  Project Description Contribution Implementation Proof Of Concept Entrepreneurship Sustainable
• Team
  Team Members Attributions Partnerships
• Wet Lab
  Experiments Engineering Success Parts Improvement Of Existing Part
• Dry Lab
  Model
• Human Practice
  Integrated Human Practices Education & Science Communication Collaborations Guidance For Cosmetics

Overview

As a team participating in synthetic biology competition, we rely heavily on good engineering and science practices. Throughout the project, we also measure our project as accurately as possible, from Imagination-Design to the whole Test-Learning of the results, recording our results at each stage. Therefore, in this part, we mainly recorded the DBTL cycle optimization process in three aspects: Molecular experiment, Product qualitative & quantitative analysis and Fermentation & Production. We managed various problems in the DBTL cycle to explore scientific research more conducive to experimental developments.

Molecular Experiment

Product Qualitative & Quantitative Analysis

Fermentation & Production

Molecular Experiments

1. The T_m Values of The Homologous Arms

1^st Iteration

Design

Considering the low success rate of restriction and insertion cloning, we employed Gibson Assembly and designed linearized vector with homologous arms and target gene primers.

Build & Test

We used the primers to carry out different PCR reactions on the vector and target genes respectively and obtained vector and target genes containing the homologous arms. Then we verified the products by agarose gel electrophoresis. After confirming that the length was correct and no miscellaneous bands, we carried out Gibson Assembly(incubated at 50°C) in the PCR instrument and transferred the products into the Escherichia coli JM109 competent cells, coated the screening plates and cultured overnight. However, no colonies grew on the plates.

Learn

Since the electrophoresis results of the linearized vector with homologous arms and the target genes had no problems, we believed that the reasons for no transformants lied in the failure of Gibson Assembly. We tried to analyze the reasons. After seeking advice from PhD. Zhang, we learned that the T_m values of the homologous arms designed by Snapgene and other software may deviate by several degrees Celsius from the experimental operation. Therefore, in order to avoid the chain-breaking and ligation failure of the homologous arms during Gibson Assembly reaction, the T_m values of the homologous arms should be ≥55°C. However, in the previous experiment, the T_m values of the homologous arms designed by us were only about 50°C, which would lead to the T_m values were lower than 50°C in the actual reaction. As a result, the homologous arms unchained themselves during the reaction and the Gibson Assembly failed.

2^nd ITERATION

Design

Since the previous T_m values of the homologous arms were too low to ensure successful ligation between the linearized vector and target genes, we redesigned the primers with higher T_m values of the homologous arms.

Build & Test

We employed a new batch of primers to reperform the PCR reaction to linearize the vector and the target genes, and carried out Gibson Assembly on the linearized products, then transferred the ligation products into the E. coli JM109 competent cells, coated the screening plates and cultured overnight, obtaining a number of transformants. After the colony PCR of the transformants, clear and accurate bands were seen from the electrophoretic gel.

Learn

Therefore, we can conclude that the T_m values of the primers need to be considered carefully when designing the primers of the homologous arms in order to obtain higher connection efficiency and avoid the self-unwinding of the homologous arms in the Gibson Assembly reaction.

2. Design of The Colony PCR Primers

1^st Iteration

Design

When constructing the gadusol producing Saccharomyces cerevisiae strains, we designed and constructed the YEP352 plasmid containing the gadusol producing genes (MT-Ox and EEVS) according to the relevant metabolic pathway. We also designed the colony PCR primers according to the target genes sequence.

Build & Test

We transferred YEP352 plasmids into the E. coli JM109 competent cells, coated the screening plates and cultured overnight. After the E. coli colonies had grown, we used the primers designed according to the target genes to underline the colonies and detect it by PCR. We found that although the PCR products all produced clear bands with consistent lengths in the electrophoretic gel, some of the screening medium did not grow any colony and we suspected that PCR appeared false positive.

Learn

After seeking advice from PhD. Zhang, we learned that the purpose of the colony PCR is not only to verify the existence of the target genes, but also to verify whether the target genes were connected to the position we designed. Thus, the gene fragments amplified by the colony PCR primers needed to contain partial sequences of the target genes and the vector at the same time. In this experiment, since Gibson Assembly used the target genes fragments to react with the linearized vector, we used primers that could only amplify the target genes fragments, which was prone to amplify the target genes fragments that were not fully combined with the linearized vector after Gibson Assembly, and then the false positives appeared.

2^nd ITERATION

Design

To solve the previous problem, we redesigned the colony PCR reaction primers that can amplify partial sequences of the target genes and the linearized vector at the same time. The new design this time helped us to verify whether the target genes were connected in the correct position of the plasmid in the newly grown colony.

Build & Test

We used the redesigned colony PCR primers to underline the colonies and carry out PCR reaction. We could see clear bands with accurate length in the electrophoretic gel and the corresponding colonies had already grown.

Fig.1 The PCR Result Using Redesigned Colony PCR Primers

Learn

Therefore, we come to the conclusion that the colony PCR primers that can amplify the partial sequences of the target genes and the vector needed to be designed separately. Subsequently, all of the colony PCR primers were designed according to this principle.

3. Primers Design

1^st ITERATION

Design

In order to improve the ability of the yeast to produce gadusol, we designed a plasmid that can make yeast use xylose. This plasmid skeleton was a low-copy plasmid (pY15) which carried three target genes, XR, XDH and XK.

Build & Test

We transferred pY15 plasmids into the E. coli JM109 competent cells, coated the screening plates and cultured overnight. After the E. coli colonies had grown, we used the colony PCR primers of XK to PCR the colonies. Surprisingly, We hardly saw the correct bands in the electrophoretic gel, but there were many transformants on the screening plates.

Fig.2 No Correct Bands Appeared

Learn

We consulted MSc.Liu, he analyzed that the colony PCR primers produced a stem-loop, or upstream and downstream primers paired with each other, which may cause the absence or multiple bands of the electrophoretic gel. He also guided us to verify whether the designed primers had these problems by Prime Primer software. We found that the primers designed before had serious pairing with each other. MSc.Liu suggested that we can appropriately lengthen the primers' length to avoid pairing.

Fig.3 The Primers We Designed Had Mutual Pairing Phenomena

2^nd ITERATION

Design

Based on the above suggestions, we redesigned a batch of primers with longer length and higher specificity. After verification by Prime Primer, we found that the pairing probability was also lower.

Build & Test

We carried out the colony PCR reaction in XK with a new batch of primers and verified accurate and clear bands from the gel electrophoresis.

Fig.4 The PCR Result Using Redesigned Primers

Learn

Since there are many repeated base sequences in yeast plasmid, it is more difficult to design the primers. With the help of primer design and verification software such as Prime Primer, we could conduct molecular experiments and get correct data feedback more easily.

Product Qualitative & Quantitative Analysis

1. Optimization Of The Product Extraction Method

1^st Iteration

Design

After constructing the yeast strains that could produce gadusol, we began to think about how to analyze it qualitatively and quantitatively. Although we hope to prepare it into sunscreen products in the form of yeast extract containing gadusol, there were too many impurities in yeast extract, so it was difficult to detect gadusol directly. Therefore, we proposed the qualitative and quantitative analysis of the products by Extraction - Spinning - Lyophilization - redissolution - HPLC.

Gadusol is a polar molecule, which is easily soluble in water and polar organic solvents. Therefore, we believed that yeast cells could be obtained by centrifugation, and then resuspended and soaked with methanol as solvent to extract gadusol from yeast cells.

Build & Test

We collected the S. cerevisiae fermentation liquid after 8 days of fermentation into 50 mL centrifuge tubes, and then centrifuged at 6000×g for 4 minutes and discarded the supernatant. The S. cerevisiae cell pellets were re-suspended with pure methanol and soaked for 10 min. After centrifuging at 10000 ×g , the supernatant was collected and the methanol solvent of it was evaporated with a rotary steamer. The concentrated extracts were dissolved with 10 mL ultrapure water (ddH₂O), collected in a centrifuge tube, freezed in a refrigerator at - 80 °C overnight, and lyophilized. The lyophilized sample was redissolved in 5 mM PBS solution, and the absorption peak was detected by HPLC at the same time as the standard.

Learn

Fig.5 The HPLC Results Of Gadusol Extracted From Nature And Gadusol Of Engineered Yeast Extracted by MeOH

According to the HPLC images, we found that the peak of the product obtained by direct extraction with methanol was very low. We speculated that the gadusol product in S. cerevisiae cells may be difficult to extract by direct extraction. Therefore, we decided to crush S. cerevisiae cells and then extract with methanol so that the product could be effectively extracted.

2^nd ITERATION

Design

We have comprehensively considered several crushing methods of yeast cells among which the ultrasonic crusher has the functions of crushing cell tissue, structure, dispersion, leaching, extraction and accelerating reaction. Using ultrasonic crushing can effectively crush S. cerevisiae cells. After crushing, methanol extraction may be used to improve the extraction amount of products.

Build & Test

Based on the previous experience, in order to fully crush yeast cells, we first adopted the crushing method of breaking for 30 s, stopping for 30 s and lasting for 30 min, and then extracted with methanol for 10 min.

Learn

Fig.6 The HPLC Result Of Gadusol Extracted By MeOH After Ultrosoniac

Through microscopic examination, we found that this crushing method could fully crush yeast cells but the time it took was so long that it had a negative impact on the ultrasonic crusher and users, and might bring some safety problems. Therefore, we decided to optimize the ultrasonic crushing conditions.

3^rd ITERATION

Design

In order to achieve the purpose of short-term and efficient crushing of yeast cells, we consulted MSC. Li. She suggested that we can appropriately shorten the time of crushing and stopping, and recommended that we use the method of breaking for 2 s, stopping for 4 s and lasting for 10 min.

Build & Test

After using this method to crush cells, we also carried out microscopic examination and compared the results. Although the total time was shortened by 2/3, there was no significant difference in the crushing results. This method was used for subsequent extraction, lyophilization, redissolution and HPLC detection.

Learn

However, we found that gadusol production was still very low, which showed that the low yield obtained had no obvious correlation with the extraction method, and the poor extraction results resulted from the low yield of S. cerevisiae after transformation. We decided to start with the synthetic route to fundamentally improve the yield of gadusol.

Fig.7 The Growth Curves Of Gadusol Producing Strains (Containing EEVS & MT-Ox Enzymes), Original Strain & Original Strain Containing Empty Vector

Fermentation & Production

1. Optimization Of The Fermentation System

1^st Iteration

Design

We inoculated the constructed gadusol production yeast strains in liquid medium for fermentation. In order to ensure that it could be applied to large-scale fermentation, we first determined whether the fermentation system was suitable by measuring the growth curve. Because we screened the yeast by using auxotroph as select marker, the medium we used was YNB + ammonium sulfate + other growth limiting factors.

Build & Test

During fermentation, we first inoculated single colonies in 20 mL fermentation medium for activation and then inoculated with an initial OD values of 0.01 in 250 mL conical flasks containing 100 mL fermentation medium for shake flask fermentation. However, we found that the strains grew poorly.

Learn

We consulted MSc.Ye，he noted that the 100 mL / 250 mL fermentation system had low dissolved oxygen and was unfavorable for yeast to grow. He suggested that we changed the fermentation system to 1/10 ratio and inoculated the activated yeast in 250 mL triangular flasks containing 25 mL medium for fermentation. However, the medium was too little in this system, which was also unfavorable for gadusol production. Therefore, we determined the fermentation system as 100 mL / 500 mL. Under the system, the OD₆₀₀ values in the platform stages reached about 15.

2. Production Optimization

1^st Iteration

Design

Although we have obtained good growth strains through optimization of the fermentation system, however, according to the results of the extraction steps, the content of gadusol obtained by introducing gadusol synthesis pathway into S. cerevisiae strains was still too low, which was contrary to our original intention to apply it to industrial large-scale production. Therefore, we had an in-depth discussion with the members of the modeling group.

S. cerevisiae uses an intermediate in the pentose phosphate pathway (HMP or PPP): sedoheptose 7 - phosphate(S7P) as a substrate and generateds gadusol through a two-step enzymatic reaction while using glucose as a single carbon source. When the yeast metabolizes normally, the vast majority of glucose enters the glycolytic pathway (EMP) rather than the pentose phosphate pathway, which results in a low supply of sedoheptose. Although we expressed the enzyme of gadusol synthesis pathway with high copy plasmids, the supply of precursor was too low to achieve a very high yield. In order to verify this view, We constructed a simple model of the metabolic pathway (Click it to see the details of model). The results showed that the generated gadusol was very low.

We began to explore whether the accumulation of sedoheptose could be improved by increasing the expression of relevant rate-limiting enzymes in the HMP pathway. However, this strategy did not get positive feedback in modeling: Although this strategy can theoretically improve the flux of the HMP pathway, increasing the expression will cause intracellular NADPH disorder because of the dependence of the rate-limiting enzymes on NADPH. In addition, this cannot fundamentally address the problem of less effective carbon sources in the HMP pathway. We began to consider the possibility of providing a more efficient carbon sources specifically for the HMP pathway.

After consulting a large number of literatures, we found xylose. The unmodified S. cerevisiae cannot utilize xylose, but as long as xylose reductase (Xyl1) and xylitol dehydrogenase (XDH, Xyl2) are introduced into S. cerevisiae, and then xylose can be transformed into xylulose-5-phosphate (X5P) through xylose kinase enzymes(XKs) carried by S. cerevisiae itself, so as to supplement S7P in yeast cells to promote gadusol production.

Build & Test

In order to obtain a yeast strain that could efficiently utilize xylose, we first used a high-copy plasmid to express XR and XDH, electrotransformed the plasmid into gadusol producing yeast competent cells, coated the plates and cultured them in a 28°C incubator for 1–2 days. Then the strains were underlined and verified by PCR, and after seeing accurate and clear bands in the electrophoretic gel, we replaced glucose with xylose in the medium and picked up single colony activation. After the activation of the strain was completed it was inoculated into 100 mL xylose fermentation medium in 500 mL conical flasks for shake flask fermentation, and the OD values of the strain was measured during fermentation.

Learn

The OD values we measured was low, which meant that the efficiency of xylose utilization by the strain was very poor. Therefore, we consulted MSC. Liu. He believed that there was no problem with our strategy of increasing gadusol production through xylose, but during molecular transformation, the higher the copy number and expression intensity, the better the results obtained might not be true. The low OD values might lead to greater metabolic pressure of S. cerevisiae due to the simultaneous presence of two high copy plasmids in the S. cerevisiae. Therefore, he suggested that we replaced high copy plasmids with low copy plasmids, or use constitutive plasmid to integrate XR or XDH into S. cerevisiae.

2^nd ITERATION

Design

Considering that the introduction of high copy plasmid limited the growth and metabolism of S. cerevisiae, and there was a certain risk of integrated expression before determining the appropriate expression intensity, we decided to relinearize the XR-XDH genes on the high copy plasmid with new primers and connect them to a low copy plasmid.

Build & Test

We electrotransformed the low copy plasmid into the competent cells of gadusol producing S. cerevisiae strains, coated the plates and cultured in a in a 28°C incubator for 1-2 days. Then the strains were underlined and verified by PCR, and after the accurate and clear bands was seen in the electrophoresis gel, the single colony activation was picked up. After the activation of the strain was completed, it was inoculated into 100 mL xylose fermentation medium in 500 mL conical flasks for shake flask fermentation. Meanwhile, the OD values of the strain was measured during the fermentation period, and the transformed yeast cells were collected. The yield of gadusol was detected to verify whether we have obtained a strain that can efficiently use xylose to produce gadusol.

Learn

We found that after changing to low copy plasmids, the growth of the strains in xylose medium were even better than that in glucose medium before transformation, and the OD values almost reached 20, which was very beneficial to the subsequent expansion of fermentation. At the same time, the HPLC results also showed that the yield of gadusol could be effectively increased by adding xylose. This proves that xylose can indeed replace glucose as an efficient carbon source for the production of gadusol.

Fig.8 The Growth Curve Of & HPLC Results Of Two Gadusol Producing Strains

So far, we have obtained a gadusol producing strain that can use xylose and be applied to fermentations in fermentators and large-scale industrial fermentation.

References

1. Jin Y S , Ni H , Laplaza J M , et al. Optimal Growth and Ethanol Production from Xylose by Recombinant Saccharomyces cerevisiae Require Moderate d-Xylulokinase Activity[J]. Applied and Environmental Microbiology, 2003, 69(1):495-503.

2. Park S H , Lee K , Jang J W , et al. Metabolic Engineering of Saccharomyces cerevisiae for Production of Shinorine, a Sunscreen Material, from Xylose[J]. ACS Synthetic Biology, 2018.

3. Osborn A R , Almabruk K H , Holzwarth G , et al. De novo synthesis of a sunscreen compound in vertebrates[J]. eLife,4,(2015-03-29), 2015, 4.

4. Kim, S. R., Park, Y.-C., Jin, Y.-S., & Seo, J.-H. (2013). Strain engineering of Saccharomyces cerevisiae for enhanced xylose metabolism. Biotechnology Advances, 31(6), 851–861.