A multi-functional system can achieve a more convenient effect, so that a plasmid with multiple devices can individually complete tasks that require plural mono-functional plasmids. According to this idea, we connected devices with independent functions, forming systems with complete functions.

In the process of synthesizing each device, the foundation for the formation of the final system had been laid. The first homologous segment was first added to the 3' end of the Panb1-crtE-AOX1 Terminator, Panb1-α factor-4CL-AOX1 Terminator and to the 5' end of the Panb1-crtB-AOX1 Terminator, Panb1-α factor-ACC-AOX1 Terminator as the vector.

The second homologous segment was then added at the 5' end of Panb1-crtB-AOX1 Terminator, Panb1-α factor-ACC-AOX1 Terminator and at the 3' end of Panb1-crtI-AOX1 Terminator, Panb1-α factor-CUS-AOX1 Terminator.

Finally, the third segment homologous to the gene on the vector plasmid was finally added at the back end of the Panb1-crtI-AOX1 Terminator, Panb1-α factor-CUS-AOX1 Terminator.

Three specific homologous segments were designed for using different PCR primers to add to different gene segments when constructing whole systems, which, in principle, ensure the experimental feasibility.

Using the most efficient in-fusion cloning principle, homologous recombinase is selected to construct the pathway. We design primers strictly in accordance with the instructions and implement the operating procedures: first double-enzyme digestion of the plasmids as the vectors, then PCR amplified gene fragments, and then gel electrophoresis to verify the vectors and conduct gel extraction for purification. Finally, the high-purity vectors and segments are connected by homologous recombinase to construct a system, and the ligated products are transformed into E.coli TOP10 F'.

Taking lycopene system as an example: firstly, the whole plasmid containing Panb1-crtE-AOX1 Terminator was used as a vector through double digestion, then the gene segments of Panb1-crtB-AOX1 Terminator and Panb1-crtI-AOX1 Terminator was obtained through PCR. Finally, the purified vector and the two purified segments were linked using homologous recombinase. The ligated products were then transformed into E.coli TOP10 F'. The cloned host was inoculated to LB plate containing AMP 100mg/mL for screening. After a single colony grew, the spot was picked for colony PCR verification. If the target band is observed, mix the bacteria solution with a 1:1 mixture of glycerin and water, and keep the seeds at -80℃.

After many attempts, we have made some achievements, but some works has not achieved the desired results, and most of the agarose gel electrophoresis strips are shorter than the expected length.

It is gratifying that the FMO dimer we built has been successful.

After many attempts and comparing the examples of success and failure, we conclude that the reasons for failure may come from these two points: 1. There are a large number of repeated segments on our system. Because all the promoters and terminators in our device are the same, it means that our target system will contain a large number of repeated fragments, which may cause functional confusion in the homologous recombinase. However, the successful FMO dimer does not contain multiple promoters and terminators. 2. The principle of the enzyme we use is different from the classic seamless cloning enzyme.

We came up with two solutions, 1. Replacement of method, using the enzyme digestion method with a certain success rate to construct pathway. 2. Replacement of the homologous recombinase.

Combining practical considerations, because we have successfully constructed the devices using the enzyme digestion method, and considering the impact of the epidemic on our progress, we chose to change the construction method, the enzyme digestion method.

We utilized the existing components, assemble each component, built the device, and then connected the devices one by one, stably, got the system, expecting to achieve our goal.

The first is to construct devices containing multiple single gene fragments. We first transferred the basic plasmids containing single gene which were synthesized by the company into E. coli for amplification. Plasmids containing the promoter Panb1 or Pynr071c became as vectors after double digestion; PCR was used to obtain the gene segments, and then double digestion with the same enzyme. The vectors that had undergone gel extraction and the purified segments are connected. Finally, the ligated product was transformed into E.coli TOP10F' and screened by ampicillin 100 ug/mL LB medium. If a single colony grows, pick the spot and verify the colony by PCR. After observing the target band, mix the bacterial solution with a 1:1 mixture of glycerin and water, and keep the seeds at -80℃.

Expand the training of the verified devices, and obtain a large number of devices needed to build the system.

Secondly, we constructed the system. The plasmid used as the vector is firstly digested with double enzymes, the fragments were obtained from previously built devices through PCR. Then the fragments were double digested with the same enzymes, similarly, the vector needed to undergo gel extraction. After the PCR fragments were purified, the vector and the fragments are connected. Same as above, the ligated product was transformed into E.coli TOP10 F', and then proceeded to screening, PCR verification and seed preservation in sequence.

First, double-enzyme digestion of the plasmid as the vector, then use PCR to obtain the gene segments of each element, then use the same enzymes to double-enzyme digest the gene segments obtained by PCR, and finally use ligase to connect the fragments with the vector. Take the construction of device: Panb1-α factor-crtE-AOX1 Terminator as an example: firstly, double-enzyme digestion of the vector plasmid containing the promoter Panb1. Then obtain the fragment from the AOX1-α factor-crtE-AOX1 Terminator basic plasmid by PCR, and use the same enzyme double enzyme digest the PCR fragments. After the fragment and the vector are purified, use Ligation ligase to connect the fragment and the vector to obtain the device: Panb1-α factor-crtE-AOX1 Terminator. Then transfer the ligation product to E.coli TOP10 F', and culture it into LB medium containing ampicillin 100 ug/mL for screening. After a single colony grows, the spot is picked and verified by PCR. If the target band is observed, mix the bacterial solution with a 1:1 mixture of glycerin and water, and keep the seeds at -80℃. The remaining 12 devices are constructed using the same method.

After obtaining all the devices, we constructed the system. simultaneously using both methods

The 1. multi-step connection.

Double-enzyme digestion of the plasmid as the vector, and then use PCR to obtain the gene fragments of each device, and then use the same enzyme to double-enzyme the gene fragments obtained by PCR. After the fragment and the vector are purified, they are ligated with the vector by ligase. Repeat the operation until the system is successfully built. Take the lycopene pathway as an example: First, digest the entire plasmid containing Panb1-crtE-AOX1 Terminator as a vector by double enzyme digestion, exposing the sticky end at the back of its gene fragment. Then obtain the gene fragment of Panb1-crtB-AOX1 Terminator by PCR, double digesting the gene fragment. After the fragment and vector are purified, ligase is used to connect with the vector to obtain a new plasmid: Panb1-crtE-AOX1 Terminator-Panb1-crtI-AOX1 Terminator. Then transfer the ligation product to E.coli TOP10 F' culture, and inoculate it into LB medium containing AMP100mg/mL for selection. After a single colony grows, the spot is picked, and the colony is verified by PCR. After observing the target band, mix the bacterial solution with a 1:1 mixture of glycerin and water, and keep the seeds at -80℃. Then use the new plasmid as a vector and Panb1-crtB-AOX1 Terminator as a fragment, and repeat the operation.

2. One-step connetion

The vector and fragment are obtained in the same way. After the fragments and the vector are purified, ligase is used to ligate multiple fragments with the vector at once. Take the lycopene pathway as an example: firstly digest the entire plasmid containing Panb1-crtE-AOX1 Terminator as a vector, exposing the sticky end at the back of its gene fragment. Then obtain Panb1-crtB-AOX1 Terminator and Panb1-crtI- by PCR, double digesting the gene fragment. The purified fragment and vector are ligated with the vector by ligase to obtain a new plasmid: Panb1-Lycopene-AOX1 Terminator. The last step is the same transformation, screening, verification and seed preservation.

All the constructed devices passed the gel electrophoresis, and all the respective bands were correct, indicating that the construction was successful.

Due to time limit, when constructing the system, the Panb1-crtE-AOX1 Terminator-Panb1-crtI-AOX1 Terminator plasmid and FMO duplex were finally successfully obtained through Method 1 only. The enzyme digestion method is more complicated and has a long cycle, so it is temporarily shelved. Method 2 failed after many attempts, and the band was shorter than expected. It may be that the homologous fragment in the connecting part of the two fragments is shorter, which affects the connection.

The partial success of Method 1 proved that our design is feasible in principle, but due to the limited time and the impact of the epidemic, we temporarily shelved the experiment but this does not mean that we will give up. After the iGEM finishes, we will continue this project until the complete system is successfully built. The failure of method 2 is also expected: inserting multiple fragments at once is "bite more than one can chew", and its success rate is destined to be low. So method 2 is only an attempt to accumulate experience and train team members. Scientific research requires our persevering efforts. We insist on our love and confidence in synthetic biology, and are willing to contribute to the development of synthetic biology

In order to facilitate the synthesis of pigments and short peptides that can be directly used for perm and dyeing, we initially chose to express all enzymes and short peptides extracellularly so that we can use related enzymes to synthesize pigments in the culture medium and can directly obtain them for perm and straightening hair. So we added α-factor as a signal peptide in front of all target genes, in order to secrete all proteins. At the same time, in order to enhance expression, we selected the strongest eukaryotic promoter that has been discovered: AOX1, to initiate and enhance the expression of downstream genes. And in order to achieve the stable existence of the target gene in yeast and form a high copy for efficient expression, we selected a suitable restriction site to linearize the plasmid to integrate into the yeast genome to ensure that it can remain stable after passaging. And we expect to achieve high copy through repeated integration. At the same time, we added His-tag at the end of the gene for protein purification and identification.

We first transfer the synthesized plasmid into E. coli for amplification in order to obtain enough target plasmid. We comprehensively considered the hidden restriction sites of different target genes, and finally selected two integration sites, BglII and SalI, to avoid the destruction of all genes. We cut the plasmid with BglII or SalI to obtain a linearized plasmid. Due to system limitations, we can only divide it into multiple tubes for single digestion.

Initially, we considered the single enzyme digestion product to be subjected to agarose gel electrophoresis, and concentration were performed while verifying. However, in next experiments, we found that the linear plasmid concentration we obtained was very low. It is attributed to the lower efficiency of gel extraction and the operation process: in the last step, repeating the experiment ddH2O elution will cause a large amount of plasmid-containing solution to remain at the bottom or wall of the ep tube; in the second elution , we cannot heat the ddH2O again, and the linearized carrier with a larger molecular weight is trapped by the purification column with lower elution efficiency, causing waste. In the end, due to various reasons, the plasmid concentration was not enough when we first performed the electroporation. Although part of the linearized vector was successfully electro-transformed in the follow-up test, and some of the yeast integrated with the target gene was obtained, the target protein was almost impossible to be detected in the follow-up identification due to the low concentration and the failure to form a high copy.

After carefully reviewing the information and asking authoritative sources, combined with the previous results, we found that at the concentration of the plasmid we extracted, overnight single enzyme digestion is completely enough to completely cut all the plasmids. This means that there are very few unlinearized plasmids in the reaction system, and there is no need to use electrophoresis to separate them. Therefore, in the subsequent experiments, we chose the method of ethanol precipitation to achieve the purification and concentration of the single enzyme digestion system. After enzyme digestion, only a small amount of products were taken for agarose gel electrophoresis verification, and the remaining products were directly used for ethanol precipitation and concentration, and finally a high concentration of linearized plasmid was successfully obtained. After electroporation, screening was performed on MD plates containing 500 mg/mL G418, and more single yeast colonies integrated with the target gene were obtained, and expression verification was performed.

We use the colony PCR method to test whether the target gene is successfully transformed into the yeast cells. The pipette tip of the picked yeast single colony is lightly dipped in a PCR tube filled with sterile water, and then filled with 1 mL of antibiotic YPD is cultured in a 2mL ep tube to realize the amplification of bacterial species while performing PCR verification, which saves time and reduces the risk of infection by reducing openings.

After the PCR verification of the colony was correct, we expanded the culture and induced expression. Since AOX1 is a methanol-inducible promoter and is strictly inhibited by glycerol, we cultured it in the initial glycerol-containing medium to expand the number of bacteria, and then transferred all of its bacteria to a culture medium with methanol as the sole carbon source. And add 0.5% of the fermentation volume of methanol every day, and after sampling and centrifugation, the supernatant was tested by SDS-PAGE. After multiple days of identification, we successfully detected some target bands. At the same time, because the molecular weight of PepACS is only 5.08 kDa, it is almost impossible to separate from bromophenol blue, and its migration is also greatly affected, so the position of the band is not accurate. In order to judge whether it was expressed or not, we first filtered the miscellaneous proteins through 10kDa ultrafiltration tube, then concentrated it with 3kDa ultrafiltration tube, and then detected it by SDS-PAGE. Judge whether it is expressed successfully by whether there is coloring near the bottom line.

Laccase GS115 4CL LOX2 ACC pepACS DsbC+pepACS supernatant SDS-PAGE detection.

Due to glycosylation modification during yeast expression, the apparent molecular weight will be slightly larger than the molecular weight of the actual protein. Preliminary detection of Laccase, 4CL, ACC bands molecular weight is about 75kDa, LOX2 is above 100kDa, DsbC+pepACS is about 40kDA, slightly larger than its actual molecular weight (Laccase: 57.01kDa, 4CL: 61.88kDa, ACC: 63.40kDa, LOX2: 102.88 kDa, DsbC+pepACS: 31.72 kDa), which are all within the acceptable range, can be considered to be successfully expressed. At the same time, due to the small molecular weight of 5-1, it is difficult to separate from bromophenol blue completely, which means than its migration is to be greatly affected. But according to the 10kDa lower part of the marker, it can be seen that it is distinguished from the blank control: there is obvious coloring, and the successful expression can be confirmed. There are irregular staining bands on the bromophenol blue line in the PepACS swimming lane. Although the molecular weight is not accurate, the successful expression can be judged according to the detection method we designed.

For the enzymes Laccase and LOX2 whose expression has been confirmed and have standard enzyme activity determination methods, we tried to identify their catalytic activity first.

For LOX2: use 27ul linoleic acid, 25ul tween 20, 8ml ddH20, 3M/L NaOH 183ul, and dilute to 50ml as a solution; take 200ul substrate, 1ml supernatant, and 1ml pH6.0 buffer for measuring absorbance changes at 234nm. And every 0.001 increase in absorbance is regarded as an enzyme activity unit. Its activity was successfully detected.

On SDS-PAGE, Laccase showed a low concentration, so it is not ideal to directly measure its catalytic activity. We were not sure about its activity status, thus, we decided to purify it with a nickel column to increase the concentration of Laccase so as to further detect the enzyme activity. activity.

It can be seen that there is a clear band at 75kDa, which is consistent with the band displayed in the supernatant, and the color of bands darkens as the concentration of the eluent increases, which further proves that the band we observed before is indeed the target protein rather than a high-expressed hybrid protein

After obtaining a higher concentration of Laccase after purification, we mixed 1mL of 1mmol/L ABTS solution with 1mL of supernatant after centrifugation, and added CuSO4 with a final concentration of 10mmol/L. The absorbance was measured at 420nm, and it was successfully detected enzyme activity. On this basis, in order to explore the optimal conditions for Laccase, we carried out further determinations.

Since the active center of Laccase is copper ion, we first studied the relationship between the supplemental copper ion concentration and enzyme activity. The CuSO4 concentration was adjusted to 9mmol/L and 10mmol/L respectively, and the enzyme activity of Laccase was measured under the same conditions.

We found that the enzyme activity is the highest when the final concentration is 10 mmol. This may be because when the concentration is too low, it cannot meet the needs of the Laccase active center. And when the concentration is too high, it affects the binding of Laccase to the substrate.

We also explored the effect of pH and temperature on Laccase activity.

pH: The data showed that the optimal pH of Laccse was acidic, so we used 100mM citric acid and sodium citrate to configure pH=3, pH=4.8 and pH=6.6 buffers respectively. Under the premise that the final concentration of CuSO4 is still 10mmol/L, 1mL of buffer is added to control the pH of the reaction system.

After comparison, we found that among the three sets of data measured, the activity of Laccase was the highest at pH=3.0. At pH=4.8, the activity drops sharply, but at pH=6.6, it is completely inactivated. As for the subsequent measurement value at pH 6.6 presenting lower than the initial value, we speculated that it is caused by the inadequate mixing of the solution at the beginning of the reaction.

Temperature: We separately kept the ABTS solution and supernatant at 10℃, 20℃, 37℃ in advance, and also kept the final concentration of CuSO4 at 10mmol/L, and mixed them before the measurement.

The analysis and comparison of the three sets of data shows that under the test conditions, the Laccase activity is highest at 20℃, and both low temperature and high temperature will inhibit Laccase activity. As for the decrease in absorbance at the late of the measurement at 10℃, it is suspected that the low temperature has a certain effect on the ABTS cation radicals, resulting in decrease in absorbance.

At the same time, during the detection process, we found that ROX1 has a relatively vague band, and we cannot confirm its successful expression based on the protein gel of supernatant. However, ROX1 is a key gene involved in the regulation of all pathways in our entire design, whose successful expression is very important to us, so we purified it through a nickel column for further verification. Fortunately, we purified the target band and confirmed that it can indeed be expressed.

SDS-PAGE verification after ROX1 nickel column purification

It can be seen that there is a band at 50-55kDa that is significantly different from the sample after washing and flow-through treatment. Its molecular weight is slightly larger than the theoretical value of 47.09kDa, but it can be confirmed within the range of the molecular weight change caused by glycosylation. ROX1 was successfully expressed. Although the gradient elution was not performed, there were many contaminated bands when the target band appeared for the first time, but according to the subsequent elution results, it can be judged that it is indeed the target protein.

However, after many times of SDS-PAGE tests, we found that there are still some proteins that have not been detected in bands, but the bacterial liquid PCR verification is correct. By analysis, we suspect that there are two reasons: one is that the extracellular expression of the protein is low, and it is unstable in the extracellular environment, and is easily degraded by proteases, etc.; the other is that the signal peptide we use is a universal signal peptide, absence of particular design for the target protein, the two do not fit and the protein cannot be successfully secreted to the outside of the cell.

After determining that some proteins cannot be expressed extracellularly through the existing system, we learned that the expression level of intracellular expression is much higher than that of extracellular expression. Intracellular proteins are more stable than they exist outside the cell, and the problem that the expressed protein cannot be detected due to signal peptide mismatch is avoided. Although it is more difficult to detect the protein in the cell, after considering various factors, we chose to rebuild the plasmid, remove the signal peptide, and try to express protein in the cell.

We designed new PCR primers on the basis of original sequence to ensure that the signal peptide is eliminated when the gene is amplified. However, because the signal peptide part of the plasmid with AOX1 as the promoter did not have suitable restriction sites, we could not use restriction enzymes to directly remove them, so we chose the method of using restriction enzymes to connect the fragments and insert after the constitutive promoter Panb1. Constitutive promoters can be used to continuously synthesize the target protein without induction.

In order to ensure the successful construction of the target plasmid, we first transformed the enzyme-linked product into E. coli for testing and amplification. After confirming that the target plasmid was obtained, we still used the same method as before, single enzyme digestion, ethanol precipitation and concentration, electrotransfection of yeast and expression.

In order to confirm the successful construction of the target plasmid, we first transformed the enzyme ligation product into E.coli, and spread it on an LB plate containing Kan resistance. Only the E.coli successfully transferred into the plasmid can grow normally. The colony PCR is used to identify whether the transferred plasmid is correct, and further sequence verification is performed to confirm that the plasmid is successfully constructed without mutation.

All the bands are identical to the theoretical lengths, which could demonstrate that these plasmid are correctly constructed and successfully transformed into E.coli, confirmed by sequencing.

Identical method is used in testing yeast after electroporation: colony PCR, confirmation, bright bands, expand cultivation.

The bright bands are identical to the theoretical lengths, which could demonstrate that this target plasmid had successfully transformed into yeast. Target genes are confirmed exist in the yeast of multiple bands, which could be the result of polluted electroporation cup.

The constitutive promoter doesn't need to be induced. To save time and unnecessary steps to reduce the risk of bacterial infection, instead of changing the culture medium, we culture our yeast continuously in the same medium with daily supply of glycerol as carbon source.

Total yeast protein extraction kit is used to extract the whole proteome of our yeast for further test because intracellular expression makes direct test of target protein in the medium infeasible. Then, we go for a purification through Nickel-affinity chromatography column, then apply SDS-PAGE to separate target protein from the large amount and various type of total protein to confirm the exist of our target protein.

Different from impure or permeate bands, the target protein located around 60kDa, bigger than the theoretical 53.96kDa but still within explainable and acceptable range of glycosylation modification. FMO could be confirmed as successfully expressed. The concentration of yeast total protein is so high that huge amount of impure protein is included during elution. But due to difference from impure or permeate bands, its dark color and consistency among several times of elution, this band could be verified as our target FMO,

Different from impure or permeate bands, the target protein located around 50kDa, bigger than the theoretical 33.42kDa but still within explainable and acceptable range of glycosylation modification. crtE could be confirmed as successfully expressed.

Different from impure or permeate bands, the target protein located around 50kDa, bigger than the theoretical 35.30kDa but still within explainable and acceptable range of glycosylation modification. crtB could be confirmed as successfully expressed.

Different from impure or permeate bands, the target protein located around 60kDa, bigger than the theoretical 55.86kDa but still within explainable and acceptable range of glycosylation modification. crtI could be confirmed as successfully expressed.

After confirmation of successful expression, we add indole as substrate into culture medium of FMO and FPP into the mixture of crtE, crtB and crtI to test whether the expressed enzyme is active in terms of color change of the culture medium. After culturing for a period of time, blue and red medium is occurred, which means we succeed in expression of active enzymes used to synthesize indigo or lycopene. Although the synthesis level of the pigments is too low to dye, still, it is one huge step for us.

From left to right:

GS115 medium with indole and FPP;

Panb1-FMO-AOX1 Terminator medium with indole;

Mixture of Panb1-crtE-AOX1 Terminator??Panb1-crtB-AOX1 Terminator??Panb1-crtI-AOX1 Terminator medium with FPP

Unfortunately, due to limited time, enzymes for synthesis and degradation of curcumin aren't tested. Neither does the synthesis of curcumin

Through intracellular experiment, we confirm that the suitability problem between the signal peptide and the space structure of our protein do exist, that is to say, even if the common signal peptide on the most popular engineering plasmid couldn't fit protein with any kind of space structure. This part of experiment rescues our experiment result by expressing enzymes which we could not detect in extracellular expression and realize the synthesis of pigments primarily.

It must be noted that the expression and synthesis level of our enzymes and pigments are too low to be applied in actual dyeing due to the shallow color of supernatant of intracellular expression after certification. Across all our experiments, we would say it is because the low copy number of our plasmid within the yeast. Due to limitation of time and culture scale, we have to pick the relatively big colonies which grow rapidly as yeast with high copy number. Without rounds of screening of gradient concentration of antibiotics, our identification of high copy number is only a suspect and not strict enough, since yeast with higher copy number will express higher level of antibiotic resistance product to grow faster in medium containing higher concentration of antibiotics. So it is possible that the strain we selected has too low the copy number to express enough amount of target protein without gradie