Difference between revisions of "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.

• Film Determination: This section introduced how we finally determinated our film, from plastic film to our final candidate. See how difficult and interesting it is!

• Calibration: This is our final step demonstrating the feasibility of NDM. See how perfect our project is!

Characterization of Optimal Conditions on Polylysine(PLL) Coated Assay

To see the details on the mechanism of polylysine adhesion, please refer to Project-CACCI.

To get the final PLL-coated protocol, check Protocols for further information.

Our original characterization of optimal conditions on polylysine(PLL)-coated assay requires several factors to get, including:

1. Best bacterial developmental interval along with recommended dilution conditions.
2. Best PLL-Coated Concentration and Pre-treatment Time.
3. Best PLL-Coated Time, given full consideration to the completion of bacterial adhesion time.
4. Best Measurement Interval, which illustrates the possible time for bacterial response on antibiotics pressure.
5. 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: Adhesion Dynamics.

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.

Figure 6: no antibiotics bacterial number variation through time with PLL

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:
\(\sigma =K\sigma _{0}\times (1-e^{-\frac{K_{\alpha}CV_{z}}{K\sigma _{0}}(t-t_{0})})\)
Constants value and details:

To know more about our modeling, please check Modeling: Adhesion Dynamics

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(-)

PLL(+,20)

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.

Let's do some crasy science this time! We'll show you how we test and select the most important part in our project, the special artificial original film!

Modeling guide us choose the candidates

In our modeling, we start with single bacteria force, analyse the interaction between bacteria and film, and propose the request of Young modulus (<1GPa) of film eventually.

And there are various film candidates come into the front.

Processing Film

The film must be kind of soft and one of the surface should have enough reflectance while another surface should have the ability to adhere to bacteria.
In fact, we don't hear any of this type of film. So we need to produce the film by our own.

Film I

Low Pressure Polyethylene

Low pressure polyethylene is soft enough, and is too soft.

We use aerosol paint cover one of the surface. That can make the other surface of the film will become a reflect surface. And we use 400ul 20ug/ml PLL coating the same surface at the temperature of 4℃ to make this surface has the ability to adhere to bacteria.

But we fail with film I in the end that we can't get interference fringes. Because the surface of low pressure polyethylene is not as smooth as we want, that will cause a diffuse reflection. Thus we can not get interference inescapably.

So we need the film becoem more smooth and more elastic, and then come out film II-Rubbers

Film II

Rubbers-Condom

If you want the material smooth enough and thick enough and elastic enough, that is, of cause, condom!

When we plan to use paint cover, the truth give us a hard hit. Because the film shrink too much when we spray paint.

And we try silver mirror reaction as replacement. But still fail Because the ammonia erosion is too severe.

Finally we find the final film candidate that we missed-Glass

Film III

Cover slip

The Young modulus of Glass is about 50GPa. But according to our adhesion assay result and modeling, there will several numbers of fringes changes in the experiment.

So we spraying it, coating it, testing it, and we made it!
The glass have a great potential to reflect, so we can get interference very easy.
With modeling guidance, we can know the relation between fringes changes and antibiotics concentration.
\(A_{0}+\frac{B_{0}}{A}=\frac{1}{\Delta N}\)

Then we want to develop a calibration with three point.

The fitting result indicate that our modeling and normalization operation was exactly correct and effective. OUR CRAZY MIND HIT OUR MODEL, AND OUR MODEL EXACYLY HIT OUR RESULT! WE LOVE SCIENCE!

That means the Processed Glass worth the name of special artificial original film!

You can detect various of kinds of material in solution, as long as you follow our standard to make the engineered bacteria. See our future NDM improvement plan at Future NDM

There is one thing to be sure is that whatever bacteria you used, you need to build calibration ruler to measure the concentration quantificationally. According to our modeling in calibration we need to confirm three constants 'A0', 'B0', 'n'.

\(A_{0}+\frac{B_{0}}{A}=\frac{1}{\Delta N}\)

That means we need to test at least three point to confirm the formula.

Establishement of the Calibration Ruler

To build a calibration ruler between chloramphenicol concentration and

interference fringes pattern.

In this case we use our E. coli BL21 with T7-OprF (BBa_K1593210), a type of bacteria in the best perfomance during genetically modification.(See our modification at Results-Permeability Improvement). According to our experiment results, we found the E. coli BL21 with T7-OprF (BBa_K1593210) was more sensitive to chloramphenicol.

And according to adhesion pre-experiment we chose the concentration range with 0.5ug/ml~5ug/ml in the experiment.

The changes of fringe number and test concentration shown below(take three pictures in a row with the time interval 20s),

Then we can fit this data with calibration formula, assume n=3(through trying different value of n get the best value of n) then get the simplified formula:

\(A_{0}+\frac{B_{0}}{A}=\frac{1}{\Delta N}\)

Then the fitting result illstruates as,

And we can get the value of unknown constants:

What an amazing result! The fitting result indicated that our modeling and normalization operation was exactly correct and effective. OUR CRAZY MIND HIT OUR MODEL, AND OUR MODEL EXACYLY HIT OUR RESULT! WE LOVE SCIENCE!

Reverification of Calibration

There may some arguement on our accuracy on calibration. So we need to conduct another experiment to reverficate our calibration ruler.

In order to test whether the calibration is correct and to confirm the coefficients, we choose two concentration as check point.

And use SPRING to measure its concentration. The real concentration and experiment results shown in table:

The result showed that the experiment concentration is EXTREMELY CLOSE to the real concentration, which means OUR NDM IS ABLE TO TELL ANTIBIOTIC CONCENTRATION IN VERY ACCURATE WAY.

By the way, DO NOT FORGET, operating our NDM only cost you 100 SECONDS.

Yes, such fast and accurate hardware detecting antibiotics, that is our NDM, ONLY OUR NDM.

Note: To get the program of our calibration analysis, please visit Software. To know more information on how we analyzed our results, please visit Modeling: Calibration. To see our final measurement report with theoretical analysis, please visit Measurement.