Basic fermentation

Our basic fermentation part derives from our local environmental problem, the outbreak of Enteromorpha along the coastline in Qingdao, so it is natural that we would eventually go back to the origin and try to solve the real world problem after validation of design concept in the lab. We aim to make use of Enteromorpha residue, where there is no trehalose left because it is easiest to extract. Therefore, all we need to do is to deal with the cellulose and hemicellulose left in the residue.

We do treat our Enteromorpha powder with enzymes first and yeast later.(the Enteromorpha powder serves as the stimulation of Enteromorpha residue in real-world situation) The successful survival of the recombinant yeast strains that can use either xylose or cellubiose as the only carbon source can fully prove the feasibility of our designed pathway.

Along with the proof of our concept, we validate the upstream pathway from real algae powder, which has exactly the same constituent as Enteromorpha residue, and we can say that our idea can apply to real-world problems!

MINI-GRE

Circuit construction

To explore the feasibility of MINI-GRE(genetic regulatory elements) by combination of promoters and terminators as we mentioned above, we designed four promoter-terminator pairs, and constructed four different report circuits for them (fig. A)

For circuit 1, we pair promoter CYC1 with terminator CYC1, which are among the most commonly used native promoters and terminators and also have a relative medium strength in yeast.[4] For circuit 2, promoter CYC1 is paired with terminator MINI. The MINIp-CYC1t and MINIp-MINIt, respectively, serves as the chosen pair for circuit 3 and 4.

For convenience, we named the“CYC1p-yECitrine-CYC1t-mStrawberry-CYC1t”as“CC”,“CYC1p-yECitrine-MINIt-mStrawberry-CYC1t”as“CM”,“MINIp-yECitrine-CYC1t-mStrawberry-CYC1t”as“MC”, and“MINIp-yECitrine-MINIt-mStrawberry-CYC1t” as “MM”,hereafter.

Fig. A the plasmid map of our circuit CC, CM, MC, MM. The CC circuit includes the commonly used native promoter CYC1 and terminator CYC1. The MM circuit includes the combination of MINI promoter and MINI terminator.

Results

For each circuit, the strength of promoters was characterized by yECitrine, a kind of yellow fluorescent protein was used to detect the output level of particular promoter, terminator or promoter-terminator pair. And the red fluorescent signal from RFP mStrawberry can represent the relative read-through efficiency of particular terminator in the circuits including the same promoter.

While monitoring the growth rate of four strains containing different expression regulatory devices, we also characterized the expression strength of the promoter-terminator pairs by detecting the fluorescence intensity of yECitrine in each circuit. As we can see in the bar chart, the ratio and relationship of the signals from 4 circuits become relatively stable at the early stationary phase, which hints that the expression level may reach the dynamic steady state at the time point 22 hours. And the results from 22 hours point also match the strength relationship of the two promoters in previous research, although we used another yeast strain here. [2, 5] Therefore, comparing with the mid-log phase data, we tend to believe that this results can reflect the true dynamic characteristics of the genetic regulatory devices, although we will research this phenomenon in our future work.(Fig. D)

So, in order to better reflect the dynamic behaviors of the circuits we tend to use data from mid-log phase during the growth process.

Fig. B The growth curve of the four strains with different promoter- terminator pairs. Error bars represent standard deviation of three biological replicates.

Fig. C The fluorescence/Abs600 in different time. Error bars represent standard deviation of three biological replicates.

Fig.D The fluorescence/Abs600 of strains with different promoter-terminator pairs, after being cultivating for 22 hours. Error bars represent standard deviation of three biological replicates.

Function Verification of promoters and terminators

Through comparing “CC” with “CM”, we can learn that the Fluorescence/Abs600 of “CM”is nearly three times of “CC”, proving that the strength of MINI terminator is higher than that of CYC1 terminator. (Fig. D)(At first we think of measuring this characteristic by comparing yECitrine fluorescence /mStrawberry fluorescence. However, considering that the fluorescence of mStrawberry is too low, we cannot expect an accurate result due to the high error rate. ) We assume that both MINI terminators and CYC1 terminators can effectively stop the transcription, so the mRNA of mStrawberry generated behind these terminators can hardly be detected, neither does the fluorescence of mStrawberry.

What’s more, the Fluorescence/Abs600 of “MM”is also nearly 3-fold compared with “MC”, proving that the strength of MINI promoter is higher than CYC1 promoter.

In addition, when we compared “MM” with “CC” which is the commonly used promoter-terminator pair, we found that the difference is nearly 6-fold. (Fig. D)

From our results, we confirmed that the MINI promoter and MINI terminator do be superior to CYC1 promoter and CYC1 terminator, not only in length but also in strength. (Fig. E)

Fig. E Length and output level of 4 different promoter–terminator combination. Error bars represent standard deviation of three biological replicates

Robustness in different host

In order to verify that the MINI-GRE is able to work with the same rule in a variety of yeast strains and expend the application range of these MINI regulatory devices, we invited other teams to repeat the same experiments in different yeast strains and experimental environment, which can also be an important part of our collaboration.

The time point of the data collection is early stationary phase of yeast growth. The yeast strains we used with Nanjing-China were Saccharomyces cerevisiae EBY100. The yeast strain used in Tianjin was synX, the yeast strain with chemical synthetic chromosome.

From the result of other colleges, we can know that our MINI-GRE can also work in other yeast strains. (Fig. F)

Fig. F The fluorescence/Abs600 of strains with different promoter-terminator pairs in the late from three teams, OUC-China, Tianjin and Nanjing-China(left to right). Error bars represent standard deviation of three biological replicates.

Transcription Level Assay by qPCR

Moreover, we run qPCR towards corresponding protein in order for a further validation of different promoter-terminator combination’s expression on a post-transcriptional level.

The result from qPCR assay shows that at the 22nd hour from setting up culture, the circuits reached the highest expression intensity and the expression level of four circuits is shown below.

Fig. G The relative transcription level of yECitrine and mStrawberry at the 22nd hour from setting up culture. Error bars represent standard deviation of three biological replicates.

However, the transcript level of yECitrine doesn’t matched the fluorescence very well. We infer that it may be caused by the experimental error of the total mRNA concentration and the little mismatch of the primers.

Luckily, we can still learn that the transcript level of mStrawberry is very low, which means the transcriptional read through of both CYC1t and MINI terminator can be overlooked; they can efficiently terminate the transcription, which confirmed the assumption before.

Conclusion

To draw a conclusion, from our experiments, we confirmed the superiority of the MINI-GRE (MINI promoter-MINI terminator pair), which can be summed into three aspects:

(1) Short but strong, which decreases the possibility of undesired homologous reorganization and provide significant output strength compared with commonly used promoter-terminator pair.

(2) Versatile,which can work in different yeast strains and provide robust function for extensive application.

(3) Well-modularited, has good modularity because of the low transcriptional read through efficiency from minimal terminator, which is an important characteristic in synthetic biology.


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