Pretreatment

1.Enteromorpha physical pretreatment.
2.Treat the residue with 0.2% H2O2 to remove the lignin.
3.Preparation of the enzymatic hydrolysis solution of Enteromorpha fiber.
4. The validation of xylose and cellobiose content after pretreatment.

Fig 1.1 Enteromorpha Powder .

Fig 1.2 Treat the residue with 0.2% H2O2.

Fig 1.3 Enzymatic hydrolysis solution of Enteromorpha fiber.

Yeast A--xylose fermenting strain

Circuit construction

We used pYC230 provided by our PI, as the backbone and constructed xylosidase gene XYL1 and XYL2 by Gibson Assembly. Gene XYL1 encoding xylose reductase (XR) which reduces D-xylose into xylitol, and facilitates xylose assimilation in yeast. Gene XYL2 encoding xylitol dehydrogenase (XDH). Xylitol is oxidized to xylulose under the action of xylitol dehydrogenase, which relies on Nicotinamide adenine dinucleotide (NAD+).

Selective Medium Assay

We transformed the pYC230nto S. cerevisiae EBY100 and got the xylose-utilizing strain successfully. We measured the growth rate of both our engineered strain and the negative control. If there is an obvious superiority of our new strain in xylose utilization, we can prove that the circuit works as our design. As a result, the curve well demonstrates that the strain with xylose-utilizing circuit has a higher OD600 than the negative control strain during the entire cultivation period. The culture of engineered yeast (EBY100 with XDH-XR) reaches stationary phases after 40 hours cultivation with an OD600 of around 2.35. While the control strain(EBY100) has an almost horizontal OD curve.

Fig 1.4

Proof of function

We use HPLC to detect the concentration changing of xylose, getting extra data to support our idea directly that our engineered yeast can utilize the xylose as carbon sources and the concentration of xylose decrease with time in our culture system.

The following chart shows the dynamic curve of xylose content of both the recombinant strain and negative control.

Fig 1.5

It is obvious that for our xylose-utilize strain, xylose content decreases as time goes, by while for the negative strain, the xylose content stays steady, indicating the disability of using xylose.

For the final goal of our project, we ultimately detected the production of ethanol. Here, we choose to use the SBA-Biosensor. Because it can detect the reaction of substrate catalyzed by the immobilized enzyme as an electrochemical signal, which supports us to describe a curve reflecting the ethanol change in the culture condition, and is able to give the output instantly and conveniently.

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

Fig 1.6

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 does produce ethanol on the basis of xylose, thus realizes our design and proves the concept of basic fermentation part.

This three-in-one chart demonstrates our evidence. The left chart belongs to recombinant strain that can utilize xylose and the right chart belongs to the negative control.

Fig 1.7

Fig 1.8

Yeast B--cellobiose fermenting strain

Circuit construction

We use pYC230 provided by our PI as our backbone and integrate cellubiose related gene CDT and GH-1 through Gibson. CDT encoding a cellubiose transporter, which assimilate cellubiose into intracellular,and GH-1 encoding β-glucosidase, which capable of hydrolyzing cellubiose into glucose.

Fig 1.9

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.

Strain construction

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

Proof of function

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).

Fig 1.10

Fig 1.11

When characterizing the growth rate of recombinant cells utilizing cellubiose with OD600, we found a platform stage in the middle of the curve suggesting probably a secondary growth. We figure out a hypothesis that the accumulation of the glucose when making use of cellubiose might be the reason underlying this phenomenon because a substitution process of the carbon sources can exactly lead to such a plateau in the growth curve. [1]Therefore, we detect the glucose content of the medium after that to validate our assumption.

Fig 1.12

Fig 1.13

The result (left) shows that glucose content in the medium reaches a peak at about 40 hours and then decrease rapidly. At around 70 hours, the content reaches the minimum and stay steady ever since. Considering the ethanol content (right) that also present a largest amount at around 80 hours, we believe that the glucose in yeast may be transformed gradually into acetic acid and ethanol participated in further metabolic procedure with acetic acid called Glucose Glycolysis, thus was turned into lipid.

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 2011_Edinburgh(http://parts.igem.org/Part:BBa_K523013) encoding INP-eYFP. We acquired INP using PCR from this part. Then we choose the constitutive 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 2016_Peking that give us much help. Monomer streptavidin is much smaller than that of wild type and is easier to be used in circuit. Based on above reasons, we designed plasmid J23106-INP-mSA-pSB1C3 (We call it JIM for short ) and transformed it into E.coli DH5α strain.

Fig.1 Electrophoresis result of expression vector. Lane 3&4 is pSB1C3-J23106-INP-mSA. Lane1&2 is pYD1-BAP, lane 3&4 is pSB1C3-J23106-INP-mSA, lane5&6 is pYC230-BirA

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 Assay

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 exists in the total outer membrane protein.

We used the Fractionation Separation to extract three components of the outer membrane 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 has the specific ability to bind INP-mSA.

Fig.2 Western blot analysis using HRP-biotin to evaluate J23106-INP-mSA(JIM) JIM expression. lane1, the outer membrane 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 prove that we do express INP-mSA on the E.coli surface successfully .

Protein function verification

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

The result proves that the function of protein was normal.

Fig 2.3 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 by plate reader. When we carried out the experiment, we met the problem that there are different incubation conditions in different paper. So we had to explore the best culture condition for the INP-mSA protein expression. Firstly, We did two sets of experiments to compare the fluorescence values of 2h incubation under two temperatures ( 0 ℃ and 30 ℃, 0℃ means incubate on ice.)

Fig 2.4 Expression level of INP-mSA fusion protein under different conditions.( I ) the single cell fluorescence values from E.coli DH5α cell expressing JIM fusion and DH5α without a plasmid 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 the Jim plasmid successfully expressed INP-MSA fusion protein. The fluorescence value increased after 2 hours incubation 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. [2]

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.(fig.1)

We first cloned birA into the yeast-shuttle plasmid pYC230. After transformation, it enables the yeast to transfer biotin to biotin acceptor peptide（BAP); pYD1 is another 5.0 Kbp expression circuit 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. By fusing our gene of interest to AGA2, so we can enable the yeast to display the biotin on the cell surface.

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 dyed engineered yeast and negative control respectively with FITC-streptavidin. Using fluorescent microscopy, we are able to observe the fluorescence of both.

Fig 2.7 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 quantify the expression of biotin compared with EBY100 by plate reader, and wonder when biotin can reach the maximum expression.

Fig 2.8 Expression level of biotin in different time point compared with negative controll.

As a result we can see the FI/abs600 of S.cerevisiae EBY100 with BAP+BirA is higher. And after 30h, the expression level of biotin achieved the highest amount.

Further, we want to know how many biotins are expressed on the surface of a single cell. Luckily, we found that we can confirm the expression of biotin and its expressing percent under Flow cytometer.

We use FITC-streptavidin to stain Saccharomyces cerevisiae and use 488nm exciting light to excite it and detect fluorescent signal at 525nm. As the result showed here, the fluorescent peak shifted towards the direction of stronger fluorescent, which means positive sample express more biotin acceptor peptide(BAP) Compared with the results of empty sample, 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%.

Fig 2.5

Fig 2.5.1 the x-mean level of FCM result

Fig 2.5.2 the CV of FCM result

Under the same CV, the mean fluorescence of INP-mSA expressing E. coli (positive) is 1.5 times higher than that of E. coli with an empty plasmid (negative).Considering the wild type E. coli(empty), the fluorescent of negative control is almost zero.(the slight fluorescent of it may come from the residue of PBS)

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.

Fig.8 (a) the growth curve of E.coli with JIM; (b) 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.

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

Adhesion 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.

Fig 2.10 The observation of confocal laser scanning microscopy(Nikon A1). The rhodamine-biotin is a solid dye, so it may have impurities in this picture.
（tips: please maximize the intensity of your screen when looking at this picture)

One step further, we want to confirm the connection between E. coli and Saccharomyces cerevisiae by FACS analysis, because this analysis method can observe in single-cell level.Unfortunately, however, we found the Beckman flow cytometry (FCM) in our university couldn’t excite our fluorescent dye rhodamine-biotin so we wereas not able to use it to catch dyed E. coli.

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.
To confirm the link between E.coli and S. cerevisiae further, we use Transmission Electron Microscopy (TEM), which can capture our strains in a microcosmic view. In the following results, the positive samples show clearly that several E.coli stick firmly to S. cerevisiae, indicating a successful adhesion between them.  In negative samples (where the yeast and bacteria have no plasmid introduced), however, there is no such connection. Actually, because of the loss of sampling point in TEM, we do not even spot the existence of Saccharomyces cerevisiae in negative control. We think that it may result from the dispersive distribution of E.coli and S. cerevisiae. Due to the limited time, we are not able to repeat our experiment and acquire data that is more convincing.  In future, we will certainly try again.

So far, the current results indicate that our first-stage adhesion platform is successfully established.

Fig.12 Observation of Transmission Electron Microscopy