Team:WHU-China/Protocol/Lab2

Protocol of algae-bacteria symbiotic laboratory

IGEM-WHU







Protocol of algae-bacteria symbiotic laboratory




I.Suspension co-culture conditions

1. culture medium of co-culture

Mix different ratios of LB medium (E. coli medium) and BG11 medium (microalgae medium), inoculate the same amount of algae, and culture under the same suitable conditions to detect changes in algae concentration.

2. Determine the optimal ratio of bacteria to algae

Four groups of different bacteria and algae ratios, each group of 3 parallel, obtained the growth curve of algae


Initial value

OD:  bacteria：algae

Three parallel each group

　

　

group

①

②

③

④

proportion

0:1

1:100

1:10

1:1

Actual absorbance

00:00.2

0.002：0.2

0.02 : 0.2

0.2 : 0.2


II. Biofilm formation and culture conditions established

Films of different suction filtration combinations were randomly prepared and cultured, and the membrane stability was qualitatively observed after 2 days.

Number
Materials
Diameter of pores/μm
ration
Effect (stability)
1
Mixed fiber membrane+blotting paper
0.45
10:1(3.5x10^7)
×
2
Mixed fiber membrane+toilet paper
0.45
10:1
×
3
Polypropylene film+Absorbent paper
0.1
10:1
×
4
Absorbent paper
/
10:1
×
5
writing paper
/
10:1
×
6
Mixed fiber membrane+writing paper
0.45
10:1
×
7
Polypropylene film+writing paper
0.1
10:1
×
8
Mixed fiber membrane
0.45
10:1
×
9
Mixed fiber membrane
0.22
10:1
×
10
Mixed fiber membrane
3
10:1
×
11
Mixed fiber membrane
0.45
20:2
×
12
Mixed fiber membrane
3
20:2
×
13
Mixed fiber membrane
3
10:2
×
14
Mixed fiber membrane
0.22
5:1
√
15
Mixed fiber membrane
0.45
5:1
√
16
Mixed fiber membrane
0.22
3:1
√
17
Mixed fiber membrane
0.45
3:1
√
18
Mixed fiber membrane(front)
3
3:1
×
19
Qualitative filter paper
/
10:1
√
20
Qualitative filter paper
/
20:2
√
21
Qualitative filter paper
/
5:1
√
22
Qualitative filter paper
/
3:1
√
23
Cotton
/
10:1
√
24
Mixed fiber membrane(reverse)
3
3:1
×
25
Cloth
/
10:1
√
26
Cloth
/
5:1
√
27
Cloth
/
20;2
√
28
Cloth
/
3:1
√
29
Mixed fiber membrane
3
10:1
×
30
Mixed fiber membrane
3
5:1
×
31
Mixed fiber membrane
3
3:1
×
32
Mixed fiber membrane
3
20:2
√
33
Mixed fiber membrane
3
20:1
×
34
Mixed fiber membrane
3
50:1
√
35
Mixed fiber membrane
3
100:1
×
36
Mixed fiber membrane
0.45
10:1
√
37
Mixed fiber membrane
0.45
20:1
×
38
Mixed fiber membrane
0.45
50:1
×
39
Mixed fiber membrane
0.45
100:1
×
40
Mixed fiber membrane
0.45
5:1
×
41
Mixed fiber membrane
0.22
10:1
×
42
Mixed fiber membrane
0.22
20:1
×
43
Mixed fiber membrane
0.22
5:1
×
44
Mixed fiber membrane
0.22
3:1
×
45
Mixed fiber membrane
3
50:1
×
46
Mixed fiber membrane
0.22
10:2
×
47
Mixed fiber membrane(front)
3
10:4
√
48
Mixed fiber membrane(reverse)
3
10:4
√


III. Establishment of standard method to evaluate the stability of the biofilm

Mixer pre-experiment: continuous stirring treatment of different membrane materials, qualitative observation of shedding

Experiment:

Mixed algae: algae 8.3*10^7, bacteria 8.3*10^8
The total volume of the mixture is about 15ml
The filtration pressure is 0.8Mpa and filtered to different filters to ensure the same filtration time.
Add 2ml BG11 medium to a small culture dish and incubate for 2 days at 26°C 4000Lux white light.
Place the filter on the splint, sandwich the inner wall of the mixing cup, add 300 or 400ml BG11 (stirring solution, maintain ionic strength, osmotic pressure, etc.)
Set 2-3 parallels for each speed or time group, set 6 speeds or time gradients
Setup program mixing
Place the remaining algae on the filter into a 15ml centrifuge tube, number A, pump the solution to a new filter, place it in a 15ml centrifuge tube, and re-use the stirred solution after number B.
The number of algae was determined by the hematocrit method or the chlorophyll method, and the drop rate B/(A+B)×100% was calculated.
Analyze and compare the data of each group to get the best speed and time of the formal stirring experiment.


IV. Screening materials for biofilm attachment

The membrane was filtered by various membrane materials into a biofilm, and after 2 days of culture, it was treated with the standard of 1.4.1.3 experimental results to obtain the shedding rate.

V. Verification of effects of our parts

Using the experimental material of 1.6 and the strength combination of 1.4.1.3, the biofilms filtered by different bacteria were treated to obtain the dropout rate of each film.