Team:WHU-China/Experiments/Lab2
The main purpose of this subgroup is to verify the effect of the parts that enhance the connection of the bacteria and algae, but due to the lack of prior technical support in this field, we spent lots of energy on the exploration method experiments. From determination of the suspension co-culture conditions of bacteria and algae, to the establishment of biofilm formation and culture conditions as well as preliminary screening of the biofilm system attachment materials. And then we established the standard of stirring-shedding rate, which realized the quantitative evaluation of the stability of the biofilm. On this basis, we completed the preliminary verification of our parts(second plasmid).
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I.Suspension co-culture conditions
1. culture medium of co-culture
2. Determine the optimal ratio of bacteria to algae
II. Biofilm formation and culture conditions established
III. Establishment of standard method to evaluate the stability of the biofilm
IV. Screening materials for biofilm attachment
V. Verification of effects of our parts
I.Suspension co-culture conditions
We use E.coli BL21 and Scenedesmus to form this symbiotic system. Although we will eventually use symbiotic biofilm, the suspension symbiotic system is the basis of biofilms. Only when the bacteria and algae are co-cultured well, then the biofilm structure can be formed on this basis. In this section, we mainly introduce the culture conditions of co-culture(culture medium) and the optimal ratio of bacteria to algae.
1. culture medium of co-culture
The medium of E.coli is LB medium, while the medium of microalgae is generally BG11 medium, we mix the two mediums in different proportions according to the teacher's suggestion, inoculate the same number of bacteria and algae and use the same Condition to co-culture them. The concentration of bacteria and the concentration of algae were simply represented by OD600 and OD750 , which can be easily tested and believable to some extent. We tested the OD everyday, and use it to determine which medium was the best.
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Figure 1 a. shows the growth curve of algae in co-culture under different medium; b. shows the growth curve of bacteria in co-culture under different medium.
From the growth curve, it can be concluded that the growth rate of bacteria and algae is best when the medium ratio is close to 7:1 (LB: BG11). The composition of this mixed medium is also very close to "simulated sewage." It is preliminarily proved that the this symbiotic system is suitable for environmental remediation and sewage treatment.
2. Determine the optimal ratio of bacteria to algae
We determined the medium conditions, but the ratio of bacteria to algae will greatly affect its growth status and compatibility. The best ratio of bacteria and algae still remains unkown. For this reason, we inoculate the original bacteria and algae in different proportions (4 groups, 3 parallels) and used the same culture conditions, the concentration of the bacteria and algae was represented by OD600 and OD750, and the growth status was judged by the respective growth curves.
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Figure 2 a. shows the growth curve of algae in co-culture under different ratios; b. shows the growth curve of bacteria in co-culture under different ratios.
We can draw the conclusion that the optimal ratio of bacteria to algae is between 1:1 to 1:10.
Conclusion: In this section, We obtained the preliminary culture conditions (LB: BG11 = 7:1) and the preliminary bacterial-to-algal ratio (1:1-1:10) of the suspension culture system.
It is worth noting that- here the OD value used in the mixed system to represent the concentration of bacteria and algae is not very accurate, although the second experiment has modified the OD value of algae. The measurement methods in the mixed system will be improved in later experiments.
Since the quantities determined by the two experiments need to be used in their respective experiments, we have finally determined a roughly reasonable range.
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Picture 1 a. our co-culture experiment; b. long term co-culture experiment.
II.Biofilm formation and culture conditions established
Since we had successfully co-culture bacteria and algae well in liquid, then we wanted to have a symbiotic biofilm. We made several attempts to obtain a biofilm.
After some failed attempts, we have established a means from symbiotic suspension system to biofilm. We decided to use suction filtration to form a biofilm from suspension system and then culture the biofilm in a culture dish.
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Picture 2 a.b.c. shows the failed experiment of biofilm formation (a: directly formation; b, c: culture in liquid medium) d.e.f.g. show the device of suction filtration and successful biofilm formation (culture in a dish).
III. Establishment of standard method to evaluate the stability of the biofilm
The instability of biofilm is a very big problem, to quantitatively tell how our part can improve the stability of biofilm, we must establish a standard measuring method—to give a interference and then detect the shedding rate of algae and bacteria.
Interference form:
After some failed attempts (use Peristaltic pump or shaker), we decided to use blender device and shear force as interference.
Blender device method is more stable, and can be quantified (by measuring temperature and speed, the shear force can be calculated) Shear force also has a physical meaning that this kind of force also acts on the biofilm on the microscopic scale.
In order to make the shear force intensity commensurate with the strength of our biofilm, we must adjust the it (through rotating speed and time) to appropriate range
Shedding rate calculation:
After obtaining the biofilm by the same method, put it onto the inner wall of the cylinder and start stirring the stirrer. After stirring, Collect algae A that remain on the biofilm and exfoliated algae B. Then count A and B separately. shedding rate “B/(A+B)×100% “can be calculated.
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Picture 3 a.b.c. show the blender and the detection of biofilm stability (using sheer force).
We used two precise methods to count the algae number instead of OD value——chlorophyll method and hemocytometer Counting method.
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Figure 3 a.b.c. use different method to illustrate the shedding rate and rotating speed.
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Figure 4 a.b.c.d use different method to illustrate the shedding rate and rotating time.
We can draw the same conclusion that- the speed is proportional to the shedding rate, and the shear force interference is most suitable when the speed is 250r/s. The time correlation is slightly smaller, so we finally chose 250r/s, 3min as our interference.
Conclusion: We focused on the algae number instead of both algae number and bacteria number. This is because it is hard to get the exact number in a mixed system(OD is a little rough and not so believable). We used this standard method to test our parts ‘ effect. Just after this establishment of standard method, we came out a brand new method to test both of their number each other. The method is called “double-wavelength method ” based on the Beer-Lambert Law. We will introduce it in page: measurement.
Ⅳ. Screening materials(track in our Ark) for biofilm attachment
Not only the stability of the biofilm itself will affect the shedding rate, but also the contact strength between the biofilm and its matrix material will. In order to eliminate the influence of this factor, we must choose a proper material. Those materials with strong durability and good water resistant performance are the candidate. We used the standard method to evaluate them.
Picture 4 shows 6 kinds of cotton cloths as candidate material.
Figure 5 shows the binding strength between each material and biofilm.
It suggested that material B and C had the lowest shedding rate and since the extraction filtration performance of B was better, we finally chose B as our basement material.
Picture 5 shows our final basement material-B.
V. Verification of effects of our parts
All the experiments above were preparing for this exciting segment! In this section, we would verify our second plasmid which aims to stabilize the bacteria-algae symbiotic system functions.
We used the material B and set the parameter as 250r/min, 3min for experiment and tested the following 6 parts: FimH-lectin-chl, lectin-Chl, dCBD-chl, FimH-lectin-Kan ,lectin-Kan and dCBD-Kan (3pathways, each one in two backbone). We transformed these parts to E.coli. ,obtained the biofilms that contain them, and tested the shedding rate separately. And we used BL21 and LB as control.
Figure 6 shows the effect of parts to improve he stability of biofilm (Chl stands for the pathway in PsB1C3, Kan stands for the pathway in Pet 28a).
It suggested that group 1 and group 6 had the best effect, the shedding rate significantly lower than control group and they had the lowest error bar. Especially group 1, the shedding rate was reduced almost a half. The other groups are all lower than control groups but not that obvious. This indicated that our binding part worked and showed it amazing potential in binging field. We felt quite pity that due to time limit, we hadn’t explored the best experiment condition, and we will optimize this in the future.
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Picture 6 a. shows that the algae are gathered by the bacteria (group 5); b. shows no algae gathered (group 8); c. shows biofilms remain in material after fierce shaking
Conclusion: So far, this subgroup have completed all the tasks. We not only verified the second plasmid and made a series of preparations for this. We also provided our project and other iGEM team with methods of co-culturing and forming biofilms. What's even more exciting is that we have established a standard method for quantitative measurement of biofilm stability in a step-by-step exploration which can be used to quantitatively assess the strength of all binding domains or binding proteins. We will introduce them in page:measurement and hardware.