Measurement

In our experiments, we established a method that can be used to quantitatively evaluate the parts such as binding domains and binding proteins. In the official part registry, binding proteins are assembled in a group, but in their characterization, most of them are characterized by qualitative or semi-quantitative methods. There is no standardized quantitative method for characterization, thus it is also impossible to compare their functions. To solve this problem, this year we have developed a device that can quantitatively display the degree of binding strength by shear force. Subsequently, we developed kinds of standard methods to analyze the shedding rate according to each situation to achieve quantitative analysis of the binding protein. Maybe this is a breakthrough from 0 to 1.

The system we have established is divided into two parts: 1. The selection of interference and the establishment of interference intensity standard; 2. The quantification of the mixed algae-bacteria symbiotic system.
"The selection of interference and the establishment of standard" has the following advantages:

1. If the same standard is used, the intensity of different binding proteins or binding domains can be quantitatively measured. The method is verified by experiments and shows repeatability, so the obtained data is comparable.
2. Since only the method of establishing standard system is specified, if the system does not apply to the standard we have established, the standard suitable for the system can be found by determined method, which greatly broadens the application range of the method, and theoretically our method can measure all the binding strength of binding protein or binding domain. This provides a standardized and quantitative means of detection for teams that will use related parts in the future.
3. The interference we selected has actual physical meaning. The shear force is the force of the actual water flow acting on the membrane, which is the force directly acting on the binding protein, so it is conducive to the subsequent modeling and mechanism analysis.
4. It can also be used to quantitatively assess the strength of other biofilms, such as the stability of biofilms formed by certain caries-related bacteria. Due to the complexity of our symbiotic system, only with the established disturbance standard we can not directly obtain the stability of the membrane, so it is also necessary to establish a method to measure the concentration of algae and bacteria in the mixed system. We analyzed, screened and tested various measurement methods for use under different conditions. This part can be used to assist the strength measurement of the binding protein and binding domain in the biofilm. It can also be used to measure the concentration of each component in a binary or even multi-co-culture system.


Therefore, our "The quantification of the mixed algae-bacteria symbiotic system." has the following advantages:
1. Various methods, which can be selected according to actual conditions
2. Expandable for measuring the concentration of each component in other binary or even multi-co-culture systems


In general, our "The selection of interference and the establishment of interference intensity standard" is divided into the following steps:
1. Randomly prepare a variety of membranes that bind to the binding protein to be tested.
2. Pre-experiment rough determines a suitable range of speed, so that most binding protein shedding rates are quite different
3. Select representative groups and perform speed gradient agitation treatment.
4. Analyze the experimental results and select the appropriate rotational speed as the unified measurement standard.


"The quantification of the mixed algae-bacteria symbiotic system" section is divided into the following steps:
1. According to the actual situation of the mixed system, select several alternative methods referring the evaluation of our methods.
2. Do pre-experiment to evaluation alternative methods.
3. Select the final method based on the pre-experiment results

"The quantification of the mixed algae-bacteria symbiotic system" result:

I. bacteria detection.

1. Ordinary CFU method need to use too many plate (72x5x5).
2. Simplified CFU method (96-well plate method) is suitable for coarse measurement of mixed system bacteria concentration. There are problems of plate drying and sampling amount determination(if the liquid is too much, the liquid layer will form, and the colonies cannot grow);the accuracy is not up to the requirement of measuring the shedding rate (the number of colonies per hole is <10, single Error caused by colony difference >10%).
3. Direct blood counting plate method is time consuming (staining problem, impurity resolution problem).
4. Photographing blood cell counting plate method requires high experimental conditions (oil mirror, CCD short focal length).
5. Centrifugal spectrophotometry can not completely separate the algae and bacteria.
6. Dual-band spectrophotometry can be tried, (pre-experimentally) select a better band, evaluate the error
7. Separation of bacteria and algae by suction filtration: Suitable for large algae, preliminary determination of feasibility, if the algae are small, it cannot be completely separated.

II. Algae measurement

1. Chlorophyll method: suitable for situations where time is sufficient and growth conditions are stable. Operation is cumbersome and environmental effect can cause fluctuations in chlorophyll content.
2. CFU method: difficult to cultivate, can not grow into "colonies".
3. Dry weight method: suitable for a large number of culture systems. If the amount is too small, it cannot be measured, and the drying process is cumbersome.
4. Centrifugal spectrophotometry is suitable for larger algae, preliminary determination of feasibility, temporarily unable to completely separate the algae, to be improved.
5. Direct blood counting plate method: time consuming.
6. Photographing blood cell counting board method: photo recording, graphic software analysis is relatively fast, but the data fluctuates greatly and requires multiple measurements.
7. Dual-band spectrophotometry: Try to choose a better band and evaluate the error (as an auxiliary measurement method, you can use the photo blood cell counting plate method to correct or evaluate the error)
8. Separation of bacteria and algae by suction filtration: Suitable for large algae, preliminary determination of feasibility, if the algae are small, it cannot be completely separated.

Attached:

The relevant experiments we have done this year:

I: Selection of interference and establishment of interference strength standard

1. Pre-experiment of peristaltic pump, shaker and mixer interference assessment
Experimental results: the interference of the peristaltic pump and shaker is unstable, and the interference intensity has no practical physical significance. (picture record is lost)
2. Mixer speed and time gradient experiment
Experimental results: The speed has a great influence on the shedding rate, and the time has little effect on the shedding rate.

II. Quantification of mixed system algae

1. Algae centrifugation method:

Experimental purpose:

Try to find the combination of centrifugal speed time, so that the algae and bacteria are primarily separated, and the concentration of algae can be measured. Experimental method: Mix different concentrations and proportions of algae, and carry out the speed gradient treatment, compare the OD value of the mixture at 680 nm and before mixing, the OD value of 1/2 of the supernatant and the original bacteria solution. If they are similar, the concentration of bacteria can be measuring

Experimental result:

Conclusion:

550-600rcf 1min centrifugation can separate the algae, but there are certain errors. This method need to be improved.

2. Small amount of CFU method Experimental principle: After diluting multiple gradients, a small amount of diluted 5-10 μL bacteria algae solution was added dropwise to the medium plate, and the bacteria algae density was calculated by the average number of colonies of a certain gradient.
Since the common plate is inconvenient to mark, there is a risk of mixing between different drops. In this experiment, a 96-well plate is used to prepare a medium plate and it works well.

Small amount of algae CFU method (Algae cannot grow as colonies on BG11 solid medium, failed)

Small amount of CFU method：
Single colonies can be obtained, which can be counted, but the accuracy cannot meet the requirements of our test for measuring the shedding rate. This method can be used for rough estimation of bacterial concentration and works well on solid plates that are added to the dish.

3.CFU method
The traditional CFU method is not suitable for large-scale measurement such as this experiment.

4. Chlorophyll method
Experimental principle: Chlorophyll in algae can be extracted with methanol, and the chlorophyll content is proportional to the number of algae.
This method works well. The first three groups in the experimental "speed gradient and time gradient exploration" are the detachment rate measured by the chlorophyll method.

5. Blood cell counting plate counting method
Experimental principle: The concentration of bacteria or algae is proportional to the number of algae in the blood cell counting plate, and the concentration can be calculated by the blood cell counting plate specification.
This method is too time consuming to count.
Experimental results: (This experiment is carried out simultaneously with the algae growth curve experiment).

Experimental conclusion: By using the blood cell counting plate method we can accurately obtain the growth trend of algae, but the error is slightly larger and needs to be repeated many times.

6. Photographing blood cell counting plate method
The blood cell counting plate is photographed to obtain photos, and image processing is performed by image J or MATLAB to obtain the density of algae.

Image J user interface

MATLAB user interface

By using this method, we can obtain the density of algae faster, and the last four groups in the experiment "Exploration of the speed gradient and time gradient" are the shedding rate measured by the modified blood cell counting plate method.

7. Pumping method
Experimental principle: Separation of algae and bacteria by different sizes of membranes
Experimental results: the separation is not complete, the reason is unknown, and the experimental picture record is lost.

8. Dual wavelength method
Experimental principle:
It is known that the concentration of bacteria and algae (c) and Abs (A) are proportional to within a certain range, A = kc and k changes with wavelength.
Within a certain range, the Abs of the algae mixture satisfies the formula A=k1c1+k2c2 (k1k2 is the absorbance of the pure solution of the algae at this wavelength)
It is planned to find two wavelengths λ1, λ2, and calculate the k value (k1-k4) of the algae at this wavelength by pre-experiment. For an unknown mixed solution, measure the A of these two wavelengths, that is, have the equations:
A1=k1c1+k2c2
A2=k3c1+k4c2
Solve the equations to get c1, c2

We found that the error of this method may be caused by the error caused by mixing and the error caused by wavelength selection.
8.1 Mixed error exploration experiment
Objective: To investigate the deviation caused by the mixing of algae and algae at each wavelength, and whether od satisfies the simple summation Experimental method: Full-wavelength scanning of suitable bacteria and algae suspension, mixing the algae liquid in equal volume, and mixing the mixture for full-wavelength scanning, comparing the theoretical value curve with the actual measurement curve.
Experimental results:

Conclusion: in proper concentration, the error caused by the mixure of algae and bacteria is extremely small.

8.2 wave length selection

Experimental purpose: find the wavelength combination with the smallest error, measure the concentration of each component of the mixed solution with the dual-wavelength method.
Experimental methods: Group 1: algae concentration gradient, group 2: bacterial concentration gradient, group 3, group 4: group 1 and group 2 corresponding gradients were mixed in proportion. Full-wavelength scanning of all samples (4*10)
Analysis of experimental results: (This part was completed by the iGEM team of Sun Yat-sen University)
The data passed the normality test, the homogeneity test of variance, and the exhaustive evaluation results of all wavelengths of 300-900 nm were obtained.
SYSU-Software helped to optimize the way to find the best wavelength: combination:
Assuming that A1, A2 have the same random error n,

Solve it and get;

So,

Let e be minimal

Calculate the e for all wave length