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

We first get the right fragments of XYL1,XYL2 through PCR then use pYC230 provided by our PI as our backbone and integrate xylosidase gene xyl1 and xyl2 through Gibson. The AGE result shows our periodical success.

After introducing the plasmid into S.cerevisiae EBY100 and construct the xylose-utilize strain successfully, 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 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 9. Xylose content of both our recombinant strain and negative control.

n 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 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.

We use pYC230 provided by our PI as our backbone and integrate cellubiose-degrading gene CDT and GH-1 through Gibson.

The bright band in the AGE result shows that we successfully get the fragments of GH-1, CDT-1 through PCR. The following experiment includes splicing the fragments onto the backbone pYC230.

Figure 11.

We introduce the plasmid into S.cerevisiae EBY100 and construct the cellubiose-utilize strain successfully 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!

Our idea has been turn into real-world practice!

Successfully constructed four sets of detection systems and imported into yeast EBY100

Figure 16.Different combinations of the Minip.Minit.CYC1p,CYC1t.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.

Successful validation of the mini system's expressive effect

Figure 17.Measured the intensity of excitation and emission of yecitrine, respectively 502nm and 532nm.The intensity of the four circuit from high to low is mm,cm,mc,cc.

Successful validation of the desired results in different laboratories and strains