Team:USTC/Results
That is so amazing! We finally made NDM this year! The results page will deliver the fresh results from us. Our results include these sections:
Permeability Improvement: In this section, we will demonstrate our synthetic bioloical base constrcuction, characterization of permeability improvement. This is the fundation of CACCI construction, as well as NDM.
Chemotaxis Modification: This section introduces results of chemotaxis engineering.
Adhesion Assay: This section explicitly explain adhesion methods, adhesion dynamics and adhesion protocols recommended for all users, which is an important part of our results.
Antibiotics Concentration Detection Demo: This section includes our initial test on our system, how we define our final methods and how we establshed protocols for users.
Calibration
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Growth characteristics
We detected OD600(nm) to identify the effect
of cheZ expression on E. coli through time.
The exogenous protein expression affects the growth of bacteria, which was indicated by slight slower grow-up in E. coli containing cheZ plasmid. In total, cheZ doesn't have obvious negative impact on bacteria grown.
SDS-PAGE
After induced by IPTG in 1mM about 4 h, cheZ was found in
SDS-PAGE gel, compared to the wild type (Top10) and bacteria without IPTG induction.
Through this result, we concluded that cheZ has been successfully expressed with the
IPTG induction.
See
protocol in Protocols: SDS-PAGE
Extraction of gene fragements
We successfully get gene fragements that are used for bacterial permeability improvement, which including,
micF and SoxS, these two antibiotic substance responding promoters were extracted from E. coli Top 10(K-12 strain) by conducting polymerase chain reaction on its genome.
SCVE, a viroporin originally from SARS virus, is synthesized from Sangon Co. ctd.
- OprF, a bigger porin compared with OmpF in E. coli, is extracted from P.aeruginosa，
PAO1 genome by PCR.
- CheZ, a chemotaxis related protein is also extracted from P.aeruginosa， PAO1 genome by PCR.
- Cas9, from 2015 part registry.
- gRNA-AcrB and gRNA-EmrE, these two fragments is for genetically modifed bacterial
transmembrane protein AcrB and EmrE to strongly block drug efflux system.
Characterization of Optimal Conditions on Polylysine(PLL) Coated Assay
To see the details on the mechanism of polylysine adhesion, please refer to Adhesion: Polylysine.
To get the final PLL-coated protocol, check Protocols: PLL Coated Assay for further information.
Our original characterization of optimal conditions on polylysine(PLL)-coated assay requires several factors to get, including:
- Best bacterial developmental interval along with recommended dilution conditions.
- Best PLL-Coated Concentration and Pre-treatment Time.
- Best PLL-Coated Time, given full consideration to the completion of bacterial adhesion time.
- Best Measurement Interval, which illustrates the possible time for bacterial response on antibiotics pressure.
- Possible Determination Interval, which refers to antibiotics response interval.
Here we present all the optimistic conditions for users to get the effective results on antibiotics using our CACCI and SPRING.
Best Bacterial Developmental Interval Along with Recommended Dilution Conditions
According to experience on E. coli development based on OD detector. We concluded that bacteria exploding during Logarithmic Period(OD:535nm about 0.4~0.5) are at the most energetic moments with proper bacterial density. Consequently, we highly recommend user to culture bacteria at Logarithmic Period and then dilute bacterial solution about twenty times to fifty times.
Best PLL-Coated Concentration and Pre-treatment Time.
Best PLL-Coated Conditions, which include PLL-Pretreatment time, PLL-Coated concentration and PLL-Coated time are measured for best adhesive condictions for bacterial treatment.
As for PLL-Pretreatment time, we adopt traditional pretreatment time, incubating about 12~16 h, for recommendation, which is originally used for neurons coated on neural science research. And when it comes to PLL-coated concentration, experience on neurons adhesion is also precious. According to previous research, PLL concentration in the interval of 20 ug/mL to 100 ug/mL is quite effective for bacterial adhesion.
Adhesion Assay with PLL treatment
Here we deliver the PLL treatment
results comparing with no PLL treatment assay. During pre-experiment adhesion assay, PAO1, a
strain of Pseudomonas aeruginosa, is used for adhesion effects.
The picture below showed the amount of bacteria(PAO1) under microscope without PLL treatment, which is abbreviated as PLL(-). To observe capability of bacterial adhesion, we wash observed place with PBS:
After elution, a significant decline in the number of bacteria is recorded. As a matter of fact, there is no bacteria in observed field after PBS washing.
To get the algorithm of bacterial counting, please refer to Modeling: Bacterial number and movement analysis.
As for treatment with PLL in 20 ug/mL, which is abbreviated as PLL(+,20), the impressive adhesion effect is observed. To ensure the exact effect, bacteria observing field is washed twice by PBS.
The number of bacteria doesn't decline, and on the contrary, we see slight increase of bacteria number. The possible reason is constant bacterial settlement during adhesion assay. After twice PBS wash, the number of bacteria is relatively stable as well. Consequently, we concluded PLL has significant adhesive ability on bacteria.
Best PLL-Coated Time
Then, we tried to figure out the cohesive effect of
polylysine through time to get the best PLL-coated time. We gathered data just after PLL
treatment(0s), after 1min and after 5min. And we also recorded bacteria number in the
following 20 s. Here we provide the analysis of bacterial number variation after PLL
treatment.We respectively use PAO1, a kind of Pseudomonas aeruginosa and HCB1, a
kind of E. coli to handle the assay. PAO1 contains self-adhesive ability and HCB1
has strong mobile ability. Using these genetically natural bacteria, we would conclude with
the effect of polylysine treatment.
As for PAO1, Without polylysine coated, bacteria have strong swimming ability. Because PAO1 is a kind of self-adhesive bacteria for experiment, thus we could see adhesive bacteria increasing during assay.
With 20 ug/mL polylysine treatement, bacterial adhesive effect due to strong electrostatic adhesion becomes stronger, and we, as well are able to observe the number of adherent bacteria gradually rising until reaching equilibrium.
After mathematical simulation we found that the adhesion rate of PAO1 by polylysine after treatment of PLL is increased, while for E. coli, the adhesion quantity rise, not fall.
As for HCB1, a kind of E. coli and the number of bacteria inside observed
field is stable and relatively declined because of its lack of capability of
adhesion.
After treated with 20ug/mL polylysine, adhesive bacteria number obviously increased:
Modeling on adhesion process strictly demonstrated the adhesion assay fits Langmuir absorption isoform.
Simulation
result:
Constants
value and details:
To know more about our modeling, please check Modeling: Adhesion Assay Modeling
This significant reverse of bacterial adhesion tendency stronly proved that polylysine has effective adhesive ability for SPRING.
After we confirmed the feasibility of PLL treatment for bacterial adhesion, then the treating time should be taken into consideration because only in the case of stable adhesion is effective for SPRING to gather effective and stable data.
As the plot illustrates, bacteria number is growing at the beginning of PLL treatment because of opening strong electrostatic adsorption derived from PLL. And after 1min, the number of bacteria become stable, and there is nearly no difference on bacteria number comparing after 1 min treatment to after 5 min treatment.
Consequently, we recommended that PLL treatment after 5 min would be a promising set for SPRING to output stable data.
Best Measurement Interval
According to chemotaxis, bacteria responsing to surrounding pressure or beneficits will be presented as change of bacteria movement. Here we analyzed bacterial movement data treating with different antibiotics concentration, specificly, chloromycetin concentration. And we gathered bacterial movement data just after antibiotics treatment(0s), after 1 min treatment and after 5 min treatment, and we again recorded all pictures in the following 18 s to get the dynamic data. Then, we concluded from the percentage of bacterial movement as an important data to intercept the effect of bacterial response on antibiotics.
The measurement assay is illustrated as following:
PLL(-)
| Antibiotics Concentration(ug/mL) | Incubation Time(s) |
|---|---|
| 0 | 0 |
| 0 | 60 |
| 0 | 120 |
| 0 | 180 |
| 0 | 240 |
| 0 | 300 |
| 1 | 0 |
| 1 | 60 |
| 1 | 300 |
PLL(+,20)
| Antibiotics Concentration(ug/mL) | Incubation Time(s) |
|---|---|
| 0 | 0 |
| 0 | 60 |
| 0 | 120 |
| 0 | 180 |
| 0 | 240 |
| 0 | 300 |
| 0.1 | 0 |
| 0.1 | 60 |
| 0.1 | 300 |
| 0.5 | 0 |
| 0.5 | 60 |
| 0.5 | 300 |
| 1 | 0 |
| 1 | 60 |
| 1 | 300 |
When bacteria are treated with PLL, the movement of bacteria is been limited for strong
adhesion, after 5 min treatment, the proportion of movement decreased through time.
However, when bacteria are not treated with PLL, incubated in 1ug/mL chloromycetin solution, we got the movement percentage of bacteria as following:
The amount of moving bacteria is relatively increasing after 5 min antibiotics treatment. Bacause of no PLL treatment, we could see the relative increase of bacteria movement percentage.
As for bacteria treated with 20 ug/mL PLL, different pattern is observed:
This is the bacterial movement percentage variation with time, treated with 0.1 ug/mL chloromycetin solution. Antibiotics pressure is not that strong, consequently, the movement of bacteria is not ignited significantly.
When it comes to 0.5 ug/mL chloromycetin solution, the proportion of the moving bacteria is
increased. And as a matter of fact, the increase is quite corresponding through time,
reflecting bacteria impressive response on antibiotics substance.
What if the concentration of chloromycetin solution up to 1 ug/mL?
A
quite linear increase of the proportion of the moving bacteria with time is observed.
Consequently, according to our experiment, genetically naturally bacteria could respond to chloromycetin solution from 0.1 ug/mL to more than 1ug/mL, which is quite promising after genetic modification.
