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 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.

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 1.11

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!

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.

E.coli strain construction

We assume expressing monomer streptavidin on the outer membrane of E.coli will help us using biotin-avidin system to link E.coli and S.cerevisiae. In part registry, we find BBa_K523013 constructed by team Edinburg encoding INP-eYFP. We acquire INP using PCR from this part. Then we choose promoter J23106 from part registry because it can express protein continuously so that we can have a relative steady co-culture system. The mSA part is from team Peking that give us much help. Monomer streptavidin is much smaller than wild type and is easier to use in circuit. Based on above reasons, we designed J23106-INP-mSA-pSB1C3 plasmid and trans-form into DH5α strain.

Figure 3.1 Electrophoresis result of expression vector. Lane 3&4 is pSB1C3-J23106-INP-mSA.

After the sequencing results confirmed that we successfully cloned the J23106-INP-mSA-PSB1C3 expression vectors, we transformed the target plasmids to DH5α for amplification.

Protein expression verification

If the protein is successfully expressed, they will be displayed on the extracellular mem-brane of the E.coli. Therefore, we can verify protein expression by detecting whether INP-mSA is displayed in the outer membrane protein.

We used the Fractionation Separation to extract three components of the outer mem-brane protein, the intimal protein and the cytoplasmic protein of engineered E.coli. Further Western Blot aim to confirm the previous result specifically and We choose to use horseradish peroxidase-biotin because it have the specific ability to bind INP-mSA.

Figure 3.2 Western blot analysis using HRP-biotin to evaluate JIM expression. lane1, the outer mem-brane of JIM; lane2, the intimal protein of JIM; the cytoplasmic protein of lane3; lane4, the outer membrane DH5α; lane5, the intimal protein of DH5α; lane6, the cytoplasmic protein of DH5α.

Western Blot result shows that there is a clear protein band at corresponding position with a correct size and the result specifically. It makes sure that we do express INP-mSA on the E.coli surface successfully .

Protein function verification

After verifying the protein expression, we want to prove that the protein can function as normal. We used Rhodamine-biotin to immunofluorescence stain the engineerd E.coli and negative strains DH5α to observe whether the bacteria had fluorescence under fluorescence microscope.

The result proves that the function of protein was normal.

Figure 3.3 Rhodamine-biotin immunofluorescence staining of engineerd E.coli(Right) and negative strains(Left).

Meanwhile, we would like to quantify the expression of INP-mSA compared with DH5α with plate reader. When we carried out the experiment, we met the problem that there are different incubating conditions in different paper. So we had to explore the best incubating condition of the INP-mSA protein expression. We did two sets of experiments to compare the fluorescence values of 2h incubation under two conditions (0 ℃ and 30 ℃).

Figure 3.4 Expression level of INP-mSA fusion protein under different conditions.( I ) the fluores-cence values of E.coli DH5α cell expressing JIM fusion and DH5α under 0 ℃ incu-bation for 2h. ( II ) the fluorescence values of E.coli DH5α cell expressing JIM fusion and DH5α under 30 ℃.

The image showed that E. coli containing Jim Plasmid successfully expressed INP-MSA fusion protein. The fluorescence value was higher after incubation of dye 2h under 30 ℃ conditions. We speculate that there may be an ice dye incubation that causes ice nucleation proteins to form crystals that inhibit the combination of fluorescent dyes and the MSA of fusion proteins.

Moreover, we want to confirm the connection between E.coli and S.cerevisiae by FACS analysis, because this analysis method can observe in single-cell level. We can use Rhoda-mine-polyethylene glycol-biotin to dye the E.coli containing JIM Plasmid for char-acterizing the expression of the protein by fluorescence detection.

Unfortunately, however, we found the Beckman flow cytometry (FCM) in our university couldn’t excite our fluorescent dye rhodamine-biotin so we was not able to use it to catch dyed E.coli. In spite of this, luckily we found that we can use FCM to confirm the ex-pression of biotin and its expressing percent. We use FITC-streptavidin to stain on S.cerevisiae and use 488nm exciting light to excite it while receive it at 525nm. As the result showed, the fluorescent peak shifted towards the direction of stronger fluorescent, which means positive sample express more BAP. Compared with the results of empty sam-ple, we can ensure that BAP is displayed on the surface of Saccharomyces cerevisiae. Above the threshold, there are 48% of Saccharomyces cerevisiae in the positive sample. While in the negative sample and empty sample, the percentage is only 28%.

Figure 3.5

Yeast strain construction

Here we need to construct the vector of BirA (biotin ligase) and BAP (biotin acceptor peptide) in yeast for displaying biotin on the surface.

We first cloned birA into the yeast-shuttle plasmid pYC230. After transformation, it ena-bles the yeast to transfer biotin to biotin-receiving peptide bap. pYD1 is another 5.0 KB ex-pression vector designed for expression, secretion, and display of proteins on the extracel-lular surface of S.cerevisiae. The vector contains AGA2 gene from S.cerevisiae encoding one subunit of the a-agglutinin receptor. Fusing our gene of interest to AGA2, so we can enable the yeast to display the biotin on the extracelluar surface.

Figure 3.6 Electrophoresis result of expression vector. Lane1&2 is pYD1-BAP, lane 3&4 is pSB1C3-J23106-INP-mSA, lane5&6 is pYC230-BirA.

After successful construction of plasmids, we introduced pYD1 containing BAP and pYC230 containing Bira into S.cerevisiae EBY100.

Protein expression verification

We want to test whether biotinylated bap is successfully integrated in the yeast cell wall. So we dye engineered yeast and negative control respectively with FITC-streptavidin. Using fluorescence microscopy we are able to observe the fluorescence of both.

Figure 3.7 The fluorescent staining of yeast with FITC-Streptomyces. (left) S.cerevisiae EBY100 without target gene; (middle) S.cerevisiae EBY100 with BAP+Bira though galactose induction for 12h; （right) S.cerevisiae EBY100 with BAP+Bira though galactose induction for 32h.

Meanwhile, we would like to quanty the expression of biotin compared with EBY100 with plate reader and wonder when biotin can reach the maximum expression.

Figure 3.8 Expression level of biotin in different time compared with negative controll.

And after Xh, the expression level of biotin achieved the highest amount.

Co-cultivation verification

We aimed to build an adhesion platform of E. coli and S.cerevisiae. Therefore, first we need to select a culture condition under which the heterogeneous cells can both grow happily. Taking into account the expression of the BAP in S.cerevisiae, galactose is required to induce protein expression. And to avoid diauxic growth of yeast when changing a new carbon source in the medium, we ultimaytely selected the YNB-CAA galactose medium. In order for the optimize expression of INP-MSA fusion protein, the in-ducing expression of BAP, and the growth rate of two heterogeneous cells, we took 28 ℃ as the culture temperature.

We characterized the growth curve of E. coli with JIM target gene and that of S.cerevisiae containing BirA & Bap under the mentioned culture condition. And the dry group wonder some parameters of the growth of E.coli and S.cerevisiae, as well as the consumption of galactose.

Figure 3.9 (left) The growth curve of E.coli with JIM; (right) the growth curve of S.cerevisiae containing BirA & Bap.

From the image we can tell, when the yeast and E.coli grows at 28 ℃, the culture time needs to be longer than 48h in order to make the yeast take a dominant proportion. We adjust the YNB-CAA galactose medium containing S.cerevisiae initial OD600 to 1.

Meanwhile, we used HLPC to measure the consumption of galactose.

Linkage validation

In order to illustrate that in practice, the co-culture of S.cerevisiae and E.coli can truly form the collaborating platform of heterologous cells, we will cultivate the two strains together by adding E.coli to the galactose-induced yeast. The co-cultured samples will then be dyed with rhodamine-biotin and FITC-streptavidin.

As is shown below, we can see that there are quite a few areas of overlapping red and green fluorescent in positive group where there are both S.cerevisiae and E.coli in the sample, indicating that they are very likely to have linked to each other. The negative control of S.cerevisiae and E.coli however, has either red or green fluorescent. And for the empty group, there is no fluorescent detected at all.

Figure 3.10 The co-cultured samples will then be dyed with rhodamine-biotin and FITC-streptavidin（tips: please maximize the intensity of your screen when looking at this picture）

Finally, we use the scanning tunneling microscope to shoot the microscopic structure of the connecting system and get a clearer picture of the connection structure. So far, we can say our adhesion platform is successfully established.

Figure 3.11 Observation of scanning tunneling microscope