Banamax Program Results
SLAC: Small laccase catalyzes was expressed and validated biologically active.
We transformed Pichia pastoris with constructed plasmids and cultured them for enzyme characterization and activity measurement.
Figure1: Different Pichia pastoris strains with different functions were cultured.
Figure1: Different Pichia pastoris strains with different functions were cultured.
SLAC Enzyme Secetion
By SDS-PAGE and silver staining, The strains transformed with SLAC exressing plasmids were detected SLAC enzyme secretion,
Figure 2 the SDS-page result shows that Pichia pastoris have successfully secrete SLAC protein into superfluous liquid.
Figure 2 the SDS-page result shows that Pichia pastoris have successfully secrete SLAC protein into superfluous liquid.
SLAC Enzyme Activity Measurement
The four kinds of signal peptides fused with SLAC were expressed and released by Pichia pastoris. Different enzyme showed different curve, the highest enzyme activity comes from SLAC fused with FLO10 signal peptide while SLAC fused with PHO1 showed delayed increase of enzyme activity.
Figure3:| Demo of enzyme activity test. The gradient color shows different enzyme activity.(The combination of secreting quantity and unit enzyme activity)
Figure4:|The enzyme activity of SLAC fused with different signal peptides, continuously recorded in 10 days.
Figure3:| Demo of enzyme activity test. The gradient color shows different enzyme activity.(The combination of secreting quantity and unit enzyme activity)
Figure4:|The enzyme activity of SLAC fused with different signal peptides, continuously recorded in 10 days.
PelA: pectate lyase from Aspergillus nigra EIM-6 was expressed and validated biologically active.
In theory, saccharomyces cerevisiae containing the pelA gene can synthesize pectinase. We use DNS method to measure the enzyme activity in bacterial solution. The data of enzyme activity are shown as follows. From the recording, we can see that the enzyme activities from five different engineering yeast increased at first and then decreased. On the third day, it reach the maximum.
Figure5. Enzyme activity curve of different signal peptides fused pelA.
Figure5. Enzyme activity curve of different signal peptides fused pelA.
VP: Versatile peroxidase was expressed and characterized
The expression and activity of the VP enzyme was measured. The experimental scheme was improved by adding heme and appropriate concentration of Mn2+, to the BMM medium and changing the PH of the buffer from 5.5 to 4.5. VP enzyme was detected by SDS-PAGE but enzyme activity cannot be detected yet, this may be resulted by mistake folding, which will be optimized in future work.
Figure6: Expressed VP was detected by SDS-PAGE
Figure6: Expressed VP was detected by SDS-PAGE
PH responding system
When selecting the response promoter of pH=5, we selected PgpdA promoter, and we tested its expression. We added the green fluorescent protein gene to the promoter and transferred the constructed plasmid into Pichia pastoris.
We cultured Pichia pastoris containing PgpdA-gfp in pH=4/5/6 medium for 4 hours, and then the fluorescence of each tube was observed under UV lamp. The results showed that it had better expression effect under the condition of pH=5.
When we selected the promoter responding pH=2, we chose the Pgas promoter. Similarly, we connected the green fluorescent protein gene to the Pgas and transferred Pgas-gfp into Pichia pastoris. The expression was induced in pH=1.6/2/2.5/3 medium. As a result, we did not observe green fluorescence. But we believe that it can be well expressed in our system, so we still add it to our entire pathway for further verification.
We cultured Pichia pastoris containing PgpdA-gfp in pH=4/5/6 medium for 4 hours, and then the fluorescence of each tube was observed under UV lamp. The results showed that it had better expression effect under the condition of pH=5.
When we selected the promoter responding pH=2, we chose the Pgas promoter. Similarly, we connected the green fluorescent protein gene to the Pgas and transferred Pgas-gfp into Pichia pastoris. The expression was induced in pH=1.6/2/2.5/3 medium. As a result, we did not observe green fluorescence. But we believe that it can be well expressed in our system, so we still add it to our entire pathway for further verification.
Characterization
In this section, we choose the part from iGEM13_Northwestern, and we do experiments to verify that if that part can be utilized by our team. We characterized it by measuring its fluorescence intensity. The part is a pH sensitive promoter called GadA-RBS (BBa_K1231001), which should be high expressed in pH=5. The faction of this part was described in detail on the part page which iGEM13_Northwestern edit. Our work is to characterization it again and find if the result we got is same to our expectation. We linked the part fragment with gfp gene by overlap extension PCR and transferred it into E. coli to express gfp gene，and cultured it in pH gradient medium. We designed five kinds of medium, which pH=3\4\5\6\7. Because there was no bacterial growth in pH=3 and 4, we abandoned the data for these two. We get the data from the plate reader and made it into a bar chart. The real result is not fit for our expectation.
Improvements
Description
Part number:BBa_K3196033
This year we have a very important part belongs to blue multicopper oxidases. We produced a new part that is based on polyphenol oxidase. This part is an improvement of an existed part BBa_K500002, which contains a Geneart Laccase that can degrade lignin with copper ion. Compered with part BBa_K500002, We added a powerful signal peptide called FLO10-αpro before the gene.
This year we have a very important part belongs to blue multicopper oxidases. We produced a new part that is based on polyphenol oxidase. This part is an improvement of an existed part BBa_K500002, which contains a Geneart Laccase that can degrade lignin with copper ion. Compered with part BBa_K500002, We added a powerful signal peptide called FLO10-αpro before the gene.
Design
We compared the two pathways to find the possible protein expression enhancement in pichia pastoris GS115.
We inserted the BBa_K500002 fragment after FLO10 signal peptide on pPIC9k plasmid using SnaBI and EcoRI. For control, we also replaced FLO10 signal peptide on pPIC9k plasmid by BamHI and SnaBI. So we can compare activity between BBa_K500002 with or without FLO10 signal peptide. Following is the DNA gel electrophoresis result of construction.
We inserted the BBa_K500002 fragment after FLO10 signal peptide on pPIC9k plasmid using SnaBI and EcoRI. For control, we also replaced FLO10 signal peptide on pPIC9k plasmid by BamHI and SnaBI. So we can compare activity between BBa_K500002 with or without FLO10 signal peptide. Following is the DNA gel electrophoresis result of construction.
Result
We transferred the constructed plasmids to Pichia Pastoris and cultured them under the same conditions for 5 days (which was the best time for expression in our main program). We measured the activity of the enzyme in a standard system. But we don’t obtain the enzyme activity data of this two part. Partly because the Geneart Laccase is wrongly folded in the Pichia pastoris GS115. Instead we break the cell by repeated freezing and thawing of liquid nitrogen and run the SDS-PAGE. Both of the constructed yeasts are cultured to the OD2.0, and treated under the same condition. Here we can find the difference protein content:
Demonstration
Major achievements:
(1) We screened out six signal peptides, and finally selected the signal peptide FLO10- α pro, which has the highest expression level, and combined it with our genes that degrade lignin and pectin to form a system for extracting cellulose from banana stalk.
(2) We fermented banana stalk in a fermentation device and obtained preliminary results in preparation for the next step of industrialization.
(2) We fermented banana stalk in a fermentation device and obtained preliminary results in preparation for the next step of industrialization.
signal peptide selection
We constructed the pathway, linked the six signal peptides to SLAC,VP and pelA, and electro transferred them into Pichia pastoris for expression. Then we first broke up the cells and verified the protein content using protein glue (figure).
Then we induced the expression of Mut+ phenotypic recombinant yeast. In SLAC, we induced the expression by adding methanol to BMM medium, and then the fluorescence intensity was determined by DNS spectrophotometry. The optimal signal peptide FLO10- α pro was screened out.
As for pelA, We failed of determination by DNS spectrophotometry at the beginning, then we changed to titration for the determination of Pectinase activity, and finally obtained the results
Then we induced the expression of Mut+ phenotypic recombinant yeast. In SLAC, we induced the expression by adding methanol to BMM medium, and then the fluorescence intensity was determined by DNS spectrophotometry. The optimal signal peptide FLO10- α pro was screened out.
As for pelA, We failed of determination by DNS spectrophotometry at the beginning, then we changed to titration for the determination of Pectinase activity, and finally obtained the results
Banana stalk degumming test.
The untreated banana stalk is dark brown, bundled with fiber, and there are a large number of sand-like and sticky substances in the middle of the fiber bundle, which make the fiber difficult to peel off and can not be used. There are also a large number of structures distributed perpendicular to the next fiber bundles in the middle of each of them, which are used to connect the adjacent fiber bundles and connect them into a network. On the other hand, the banana stalk treated by us not only lost the connection structure between the fiber bundles, but also almost all the viscous particles filled between the fiber bundles were degraded. The fiber is transparent and white and can be separated.
We could clearly see that the fiber without degumming are clustered together and difficult to separate. (Fig.1). And the degummed fiber are loose. (Fig.2). At a higher magnification, we could see that there are many impurities on the fiber without degumming, including pectin, lignin, etc. They are attached to the fiber, as shown in figure 3 (the brown part is impurities). These impurities make the fiber look very dirty and they greatly affect the properties of the fiber. It can be seen more clearly under a transmission electron microscope (Fig.5, 6), with all the impurities clustered.
After biological degumming, we can clearly see that almost all the impurities attached to the fiber are removed, and the whole fiber is clean and transparent under the optical microscope (Fig. 4). This is also confirmed by transmission electron microscopy (Fig.7, 8). This shows that our engineering yeast can secrete enzymes to degrade lignin and pectin, and can be used on a large scale.
Figure7: A pilot test of banana stalk degumming with Banamax
We could clearly see that the fiber without degumming are clustered together and difficult to separate. (Fig.1). And the degummed fiber are loose. (Fig.2). At a higher magnification, we could see that there are many impurities on the fiber without degumming, including pectin, lignin, etc. They are attached to the fiber, as shown in figure 3 (the brown part is impurities). These impurities make the fiber look very dirty and they greatly affect the properties of the fiber. It can be seen more clearly under a transmission electron microscope (Fig.5, 6), with all the impurities clustered.
After biological degumming, we can clearly see that almost all the impurities attached to the fiber are removed, and the whole fiber is clean and transparent under the optical microscope (Fig. 4). This is also confirmed by transmission electron microscopy (Fig.7, 8). This shows that our engineering yeast can secrete enzymes to degrade lignin and pectin, and can be used on a large scale.
Lignin degradation ration
Lignin is one of the most abundant impurities in fiber, and the degradation of lignin is difficult. Therefore, we use the relevant enzymes to help us degrade lignin. We determined the degradation rate of lignin after pretreatment, chemical treatment and chemical and enzyme treatment under the same conditions.
We burned the fiber and measured the lignin content of ash. The experimental result show that the combination of chemistry and enzyme can better degrade lignin. Based on this result, we can believe that the enzyme-treated fiber contains less lignin.
We burned the fiber and measured the lignin content of ash. The experimental result show that the combination of chemistry and enzyme can better degrade lignin. Based on this result, we can believe that the enzyme-treated fiber contains less lignin.
