Human Practice + Experiment + Model
Field Investigation
We investigated the current environment and governance results of Taihu Lake, did a questionnaire
on
the influence of cyanobacteria bloom and finally found that the problem still existed and needed to
be
figured out in a more cost-efficient way !
Government Association
We asked Huishan Ecology and Environment Bureau for basic statistics and their guidance. We knew
that current traditional burning method was defective. Considering the secondary pollution and costs,
what we needed to do is to fix the problem of
cyanobacteria gathering in a biological way.
Pre-experiment: Cyanobacteria Degradation
Equal amount of sophorolipids were powdered to three groups of cyanobacteria with different
concentrations and mixed up to a final low content of 20 mg/L. Then we observed the degradation
effects of the
cyanobacteria.
Result:
The cyanobacteria can be degraded efficiently by sophorolipid in a short time.
Fig1. Degradation effect of cyanobacteria after 5 minutes
Promoters Screening
We had obtained 14 different promoters from Starmerella bombicola. To measure their expression
intensity, the green fluorescent protein (GFP ) gene was used as the reporter gene and expressed
by
different promoters in S. bombicola.
Fig2. GFP expression cassette
Result:
Then through the analysis of fluorescence and transcription level, we successfully defined the
intensity of different promoters. (Fig.3 and Fig.4) Ptef1 is the
strongest promoter.
Fig3. FITC-Mean of promoters Fig4. RT-qPCR of promoters
Construction of CRISPR/Cas9 System
The strongest promoter Ptef1 was used to express Cas9 protein and
sgRNA. A self-excising hygromycin
marker was also added to help find the positive transformants and remove resistance after
gene-editing. Then the Cas9 expression cassette was integrated into the genome of S. bombicola.
Fig5. Cas9 expression cassette
Result:
The verification experiments of single, double and triple gene-editing efficiency were carried out
to
edit PXA1, GFP and LEU site. Single gene-editing efficiency can up to 100%,
double one can up to 99%
and triple one can up to 30%. After the induction by galactose, the engineered yeasts can excise
hygromycin resistance gene.
Fig6. The verification experiments of gene-editing efficiency
Production of Pure Lactone/Acid Sophorolipid
Three strong promoters were used to over-express UDP-glucosyltransferase B (UGTB ) (Fig.7) to
increase
the yield of both lactone and acid type sophorolipids. Based on this, the pure acid sophorolipid can
be produced by knocking out SBLE (Fig.8), while the pure lactone one can be produced by using
strong
promoters to over-express SBLE.(Fig.9)
Fig7. Over-expression of UGTB by using strong promoters
Fig8. Knock out SBLE
Fig9. Over-expression of SBLE by using the strong promoter
Pgki
Result:
After over-expressing UGTB, the yield could be up to about twice than that of the control.
Pure
acid
sophorolipid could be produced via knocking out lactonase (SBLE ) gene, and pure lactone one can
be
produced via using a strong promoter to over-express lactonase (SBLE ).
Fig10. The yield of sophorolipids produced by different strains
Fig11. The sophorolipids production
(Left: Lactone type; Right: Acid type)
Market Analysis
Initially, we wanted to manufacture pure acid/lactone sophorolipid to tackle cyanobacteria bloom.
But
now, considering the different useful characteristics, we changed our target and wanted to use
varied
ratios of lactone and acid sophorolipids to get a better environmental protection result and explore
wider application objectives.
General Direction of Modeling
Under the background of COVID-19, in order to reduce the number of trial and error in the
experiment,
we set up Linear Pathway Model based on the idea of ‘Quantitative experiment to qualitative Model to
Quantitative experiment’.
Step 1: Experiment Data Processing
Firstly, the promoter data at transcriptional and translational levels are normalized and weighted,
and
the promoter equivalent parameter μ is obtained by using the idea of grey box model.
Step 2: Model Simulation
Then, we used Michaelis equation and the law of conservation of mass to establish Linear Pathway
Model. X4 and X5 are acid-type and lactone-type sophorolipids respectively, so we added the promoter
equivalent parameter μ to reaction 4 and reaction 5 respectively to express the promoter regulation
of
the reaction.
Step 3: Standard Curve
Next, we can select several combinations of promoters for experiments, and then fit the
experimental
data and simulated data into a standard curve, so as to determine the combination of promoters
needed
for the ratio we want.
Simulation Result
Finally, we calculated the ratio of acid-type sophorolipid to lactone-type sophorolipid by simulation
result of 225
combinations. The numerical values in the heat map represent the ratio of acid-type sophorolipid to
lactone-type sophorolipid in the simulation. According to the trend simulated by the qualitative
model, the
several combinations of promoters would be selected for experimental verification, and then a
‘standard curve’ could be drawn, which could correspond to the simulated data. In the future,
according to the trend of simulated data and the mapping relationship of real data, we can choose
the
several appropriate promoter combinations to achieve the desired ratio.
Find the Best Ratio
The pure sophorolipids were mixed to different ratios and added into equal amount of cyanobacteria.
The degradation efficiency was measured by preliminary centrifugation observation and chlorophyll
content test of living cells.
Result:
The lactone : acid sophorolipid ratio of 8:2 has the highest efficiency on cyanobacteria degradation.
The best
ratio: Lactone:Acid=8:2.
Fig12. Preliminary
concentration observation
Fig13. The degradation
efficiency of cyanobacteria
Application Test
We visited Professor Zhou who had some experience in Taihu problems in Jiangsu University to
explore
the application methods and cooperated with an Algae-Water Plant to further test the degradation
result.
Industrial Production Exploration
We visited EVONIK which had done some researchers on sophorolipid to explore the possibility to put
it into industrial production and we made sure that our product was safe by interacting with their
manager. Besides, we also did some interviews on our potential users to know their opinions.
Future Work：Construction of strains producing desired ratios of
sophorolipid
The promoters of UGTB and SBLE were replaced by other pairs of promoters to control the
ratio of
lactone/acid sophorolipid.
Fig14. The expression fragments of UGTB by using different promoters
Fig15. The expression fragments of SBLE by using different promoters
