Circuit construction

We use pYC230 provided by our PI as our backbone and integrate xylosidase gene xyl1 and xyl2 through Gibson. Gene XYL1 encoding xylose reductase (XR),which reduces D-xylose into xylitol, facilitated xylose assimilation in yeast, and gene XYL2 encoding xylitol dehy-drogenase (XDH) .Xylitol is oxidized to xylulose under the action of xylitol dehydrogenase, which relies on NAD.

Strain construction

We introduce the plasmid into S.cerevisiae EBY100 and construct the xylose-utilize strain successfully. We measured the growth rate of both our recombinant strain and nega-tive control. If there is an obvious superiority of our new strain in xylose utilization, we can prove that the strain is constructed correctly. As a result, the curve 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.

Fig 1.4

Proof of function

We use HPLC to detect the changing concentration of xylose, getting more data to support our idea directly 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.

Fig 1.5

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 xy-lose.

For the final goal of our project, we will ultimately detect the production of ethanol. Here we choose to use the SBA-Biosensor. Because it can convert the reaction of immobilized enzyme to electrochemical signal, which supports us to describe a curve reflecting the etha-nol change in the culture condition, and is able to give the output instantly and convenient-ly.

The following chart shows the ethanol content of 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 do produce ethanol on the basis of xylose, thus realizes our de-sign 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

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 introduce the plasmid into S.cerevisiae EBY100 and construct 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 guess that the accumulation of the glucose when making use of cellubiose might be the reason lying beneath because a substitution process of the carbon sources can exactly lead to such a plateau in the growth curve. 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 reach a peak at about 40 hours and then decrease rapidly. At around 70 hours, the content reach the minimum and stay steady ever since. Considering the ethanol content (right) that also present a largest amount at around 80 hours, we guess that the glucose in yeast was transformed gradually into ace-tic acid and ethanol participated in further metabolic procedure with acetic acid called Glu-cose glycolysis thus was turned into lipid.