Team:FAFU-CHINA/Measure
soil samples
We took rotten soil near the School's Zhonghua Garden and sugarcane base as soil samples for the separation of wild bacteria capable of cellulose degradation. After sampling, 5g of two kinds of soil samples were taken and mixed with 100ml 0.85% NaCl solution, which was placed in a shaking table for 30min and an ice bath for 2min. The supernatant was taken for gradient dilution to obtain soil samples diluted 10-2, 10-3, 10-4 and 10-5 times. 10-3, 10-4 and 10-5 times of soil samples were coated on the screening medium (CMC-Na medium) and cultured in an incubator at 28℃.
Fig. 1 soil sample processing steps
We counted a total of about 12 plates, two days after the culture of bacterial growth, more than half of the plate culture medium bacteria growth is good, suitable for further experiments.
Plan A
After our first discussion, we decided to select the single colony into the EP tube containing 1ml CMC-Na liquid medium. After the liquid in the EP tube becomes cloudy, we want to dip the bacteria liquid into the middle of the Congo red medium plate with a filter paper. If there is a hydrolytic ring, it will be the desired strain. The results showed that the growth rate was extremely slow, and there was still no growth two days later (the bacterial fluid became cloudy 7 days later).
Fig. 2 The steps of Plan A
Plan B
So we decided to use the second method, we used a pipette to pick the more obvious single colony on the medium, and transferred to the new solid screening medium plate (CMC-Na), two plates for every single colony.
Fig. 3 The steps of Plan A
Therefore, we selected about 57 strains three times, a total of 114 plates, all of which had bacterial growth, and some of the strains grew well. The picture shows the colony morphology of the strains we picked.
Fig. 4 The colony morphology of the selected strain growth
After that, we prepared 0.2% Congo red staining reagent and carried out Congo red staining experiment on 57 strains. We directly added 1ml prepared Congo red reagent to the plate with bacterial colonies and left it to stand for half an hour. After that, the Congo red reagent was poured out, and the plate was cleaned several times with double steaming water, and the hydrolytic ring was carefully observed. Among the 57 strains, we observed that the hydrolysate circles of 11 strains were obvious, while the hydrolysate circles of other strains were not obvious or no transparent circles were observed. Therefore, we selected 11 strains with the obvious phenomenon and good colony morphology and no contamination by miscellaneous bacteria to continue the experiment.
Fig. 5 Colonies stained with Congo red
We selected 11 single colonies and selected them to a new CMC-Na solid medium plate. Each colony was repeated, and a total of 22 colonies were cultured in an incubator at 28℃ for the first colony purification. The staining experiment of Congo red was carried out after the growth of colonies. We repeated the purification of these 11 strains for three times and did the Congo red staining experiment after each purification. However, the result of the transparent circle after the second staining was less obvious than that of the first time, and a large number of colonies after the third purification showed no transparent circle, which may be caused by the change of the culture medium formula.
The 11 strains were selected into a test tube with 5ml CMC-Na liquid medium and placed in a shaking table at 28℃ for two days. However, no bacterial liquid was obtained after repeated twice. The 11 strains were then selected into a test tube with the liquid medium of 5mlLB and placed in a shaking table at 28℃ for culture to obtain the bacterial liquid.
We took 900 ul bacterial solution from the test tube and added it to a 60% glycerol solution of 300 ul for bacteria preservation. A total of 55 tubes of bacteria preservation were stored in a 1.5mL centrifuge tube and stored in a -20℃ refrigerator. The 400UL samples were sent to the company for sequencing with 27F and 1492R.
phylogenetic tree
After getting the sequence, we use the Maximum Likelihood Method (ML) to construct a phylogenetic tree, which is a method based on nonlinear spectrum estimation; calculate the likelihood function for all possible phylogenetic trees, and then select the one with the largest likelihood function value The phylogenetic tree model is the best choice.
As shown in the figure, we measured the 16S_rDNA sequence of the bacteria with 27F and 1492R primers to identify the different species of bacteria and divide them into six subclasses I, II, III, IV, V, VI. According to the results of phylogenetic tree cluster classification, it can be inferred that they may have a common ancestor.
The numbers on the branches mean the reliability percent of Bootstraps value basedon1000replication; The branches of different classes were painted with different colour（Red branches represent the first category, black branches represent the second category, golden branches represent the third category, green branches represent the fourth category, grey branches represent the fifth category, and blue branches represent the Sixth category）, Different colours and symbols represent different species, and each arc of different colours represents a different subclass (Class I~Class VI).
Fig. 6 The phylogenetic tree of the 11 bacteria