Enteromorpha pretreatment

We treat the Enteromorpha powder (residue) with 0.2% H₂O₂ to remove the lignin then cellulase and xylanase to produce xylose & cellubiose.

Pretreatment validation

After that we detect the existence of them with HPLC. The peak appears at the same point suggesting that they are the same substance. In other words, we successfully proved that the downstream product of Enteromorpha powder after pretreatment contains mainly xylose and cellubiose.

Yeast A that ferment xylose

After introducing the plasmid we constructed that contains gene XYL1,XYL2 into S.cerevisiae EBY100. We creat a xylose-utilize strain. We measured the growth rate of both our recombinant strain and negative control to prove the superiority of our new strain in xylose utilization.

We cultivate the EBY100(XDH-XR) and the EBY100(control) in SC adding 2% xylose as the sole carbon source, adjusting initial OD₆₀₀ about 1.2，and placing in the shaking incubator of 30℃，180rpm to ferment.

The following result can well demonstrate that the strain that carries our plasmid grows much better than the strain that not and reach the stationary phases after 40 hours’cultivation.

Figure 1.8 Growth curve of strains of our recombinant strain and negative control.

For an immediate evidence, we need to know exactly how xylose content change in the medium. We use HPLC to detect the changing concentration of xylose, getting more data to support our idea that our cells can utilize the carbon sources as long as the concentration of xylose decrease with time.

The following chart shows the xylose content of both our recombinant strain and negative control. It is obvious that in our xylose-utilize strain, xylose content decrease as time goes by while for the negative strain the xylose content stays steady, indicating the disability of using xylose.

Figure 1.9 Xylose content of both our recombinant strain and negative control.

In addition, to finally realize our design, the yeast need to ferment on xylose only. Therefore, we use the SBA-Biosensor to detect the ethanol content in the medium, which can convert the reaction of immobilized enzyme to electrochemical signal and help make a curve reflecting the ethanol change in the culture condition.

The following chart shows the ethanol content of both our recombinant strain and negative control.

Gladly, the curve of xylose-consuming strain goes gradually up along with cultivating hours, and reach the plateau at around 90 hours, which is consist with the xylose consuming curve, indicating that our strain do produce ethanol on the basis of xylose, thus realizes our design and proves the concept of basic fermentation part.

Figure 1.10 Ethanol content of both our recombinant strain and negative control.

In the same way, we conduct a series of experiment to confirm that our cellubiose-utilizing pathway also worked.

For the cellolose plasmid, we integrate cellubiose-degrading gene CDT and GH-1 into pYC230 by Gibson.

Figure 1.11

We introduce the plasmid into S.cerevisiae EBY100 and construct the cellubiose-utilize strain successfully and then test the growth rate of both our recombinant strain and negative control.

We cultivate the EBY100(CDT-GH1) and the EBY100(control) in SC adding 2% cellubiose as the sole carbon source, adjusting initial OD₆₀₀ about 1，and placing in the shaking incubator of 30℃，180rpm to ferment.

Using HPLC we detect the changing concentration of cellubiose. The result shows that the concentration of cellubiose decrease with time, indicating a well utilization of cellubiose.

The following chart shows of both our recombinant strain (left) and negative control (right).

To examine the production of ethanol, we use the SBA-Biosensor, the same as in the xylose pathway.

The following chart shows the cellubiose content, ethanol content, and growth rate of both our cellubiose recombinant strain (left) and negative control (right).

We acquired similar trend of those curves except one, the ethanol content, which dropped from around 70 hours. Considering the difference between monosaccharide and disaccharide, we suppose that the accumulation of the glucose when making use of cellubiose might be the reason lying beneath. Therefore, we detect the glucose content of the medium after that to validate our assumption.

The result shows that glucose content in the medium reach a peak at about 40 hours and then decrease rapidly, which is consist with our expectation that the glucose in yeast was transformed gradually into acetic acid. Ethanol might then participate in further metabolic procedure with acetic acid thus was turned into ethyl acetate when glucose accumulates to a particular concentration.

Despite that, we can tell from the distinct difference between cellubiose-consuming strain and negative control that our approach of using yeast to transform Enteromorpha residue into ethanol finally worked!

And for a more visualized display, we use plate streaking to show the growth condition of our two recombinative strains and negative strains in different carbon source. We make three plates with either xylose, cellobiose or xylose plus cellobiose as sole or compound carbon source, respectively and monitor the growth of each strain in three different plate after cultivating for 32 hours at 30℃.

We successfully confirmed in lab through simulation that our idea has been turn into real-world practice!

Circuit construction

Figure 2.1 The plasmid map of our circuit.

We synthesized minip, minit, cyc1p, cyc1t and built four circuit with Gibson assembly by arranging them in different orders. The plasmids were then imported into yeast EBY100.

Function verify

We characterize the strength of the MINI system by detecting the fluorescence intensity of the yeCitrine. We measured the growth curves of four strains containing different expression systems, and measured the intensity of excitation and emission of yeCitrine, respectively 502nm and 532nm.

For convenience, we named the “Pmini-yECitrine-Tcyc1-mStrawberry-Tcyc1” as “mc”, “Pmini-yECitrine-Tmini-mStrawberry-Tcyc1” as “mm”, “Pcyc1-yECitrine-Tcyc1-mStrawberry-Tcyc1” as “cc” and “Pcyc1-yECitrine-Tmini-mStrawberry-Tcyc1” as “cm” hereafter.

Figure 2.2 The strength of the MINI system.

The fluorescence intensity is: mm> cm> mc> cc, in which mm circuit has the highest expression, proving our successful construction of MINI system. To draw a conclusion, the system has a strong expression, but a very short nucleotide sequence. In this example, the combination of weak promoter + strong terminator is better than that of strong promoter + weak terminator.

With the help of the other two teams, we completed the repeated testing of the MINI system. The time of the test data is the late logarithmic phase of yeast growth. The yeast strains we used with NJU-China were Saccharomyces cerevisiae EBY 100. The yeast strain used in TJU China was Synthetic yeast synX.

Figure 2.3 MINI system fluorescence measurements from three teams, OUC-Chian,TJU-China, NJU-China (left to right).

Through the fluorescence intensity map, we have not yet clear the true advantage of MINI system. But from the following figure, we can directly observe the relationship between a nucleotide base number and fluorescence intensity. The red arrows indicate the short and strong features of the mm gene circuit.

Figure 2.4 Promoter and terminator of the nucleotide base length and intensity(For example, mm represents the base length of Pmini + Tmini).

In order to verify that the MINI system was able to work in a variety of yeast strains, we invited other teams to conduct a repeat experiment. Especially in TJU-China, the use of their own synthesis of yeast to complete this experiment also verified the MINI system with versatility.

Validation of expression on transcription level

The result of qPCR shows that at 22^nd hour the system reached the highest expression intensity and the expression of four circuits is shown below. The error bars indicate s.d. of mean of experiments in triplicate.

Figure 2.5 The expression of four circuits at 22^nd hour.

In the chart, the RNA content of yECitrine comes as the following order: mm>mc>cm>cc, which is not completely consist with the result of protein level. mm’s RNA content is several times that of cc. Compared to Figure b, this difference in RNA content does not reflect the protein content very well, yet still, our MINI system has an obvious superiority over normal combination (cc), which confirmed our hypothesis. As shown above, generally the magnitude of leakage is over 100 times smaller, so it can be ignored to some extent.

To draw a conclusion, mm has the smallest size and the strongest expression with a leakage that can almost be ignored. What’s more our system functions well in different strains and experimental conditions, which proves its potential to apply in various situations.