CN111849808A - Methods of enrichment culture, inoculation and PCR amplification of PHA-producing bacteria from marine sources - Google Patents

Abstract

The invention relates to a method for enriching, culturing and inoculating marine PHA (polyhydroxyalkanoate) producing bacteria and amplifying genes by PCR (polymerase chain reaction). The inoculation method comprises the following steps: mixing marine sediments with an MM1 culture medium in a ratio of 1:100, and fully shaking and culturing in a shaking table for one period, wherein a mixture of the cultured sediments and the culture medium is called an enrichment culture solution; inoculating an enrichment culture solution in the current MM1 culture medium into the next gradient MM2 culture medium in a ratio of 1:100, and so on, completing enrichment culture for 6-8 cycles, wherein the cycle is 7-10 days; the method comprises the steps of conducting primary separation after enrichment is completed for 2-3 cycles, conducting colony PCR amplification on the purified single colony phaC synthetase gene, then utilizing agarose gel electrophoresis to separate PCR products, judging whether PHA is produced by the strain according to whether obvious bands exist on gel, utilizing PCR amplification 16Sr RNA gene, and identifying the classification status of the strain through first-generation sequencing.

Description

Methods of enrichment culture, inoculation and PCR amplification of PHA-producing bacteria from marine sources

technical field

The invention relates to the technical field of bacterial enrichment culture and PCR amplification of genes, in particular to a configuration method, inoculation method, and polymerase chain reaction (PCR) amplification of a marine-derived polyhydroxyalkanoate bacterial enrichment medium A method for 16Sr RNA gene sequence and a method for amplifying the phaC synthase gene.

Background technique

Polyhydroxyalkanoates (PHAs) are linear polyesters composed of a class of 3-hydroxyalkanoates synthesized by bacteria. This material has good biocompatibility, biodegradability and thermal processing properties of plastics, and is currently a hot material in the field of biomedical materials and biodegradable packaging materials.

The substrate specificity and synthesis efficiency of the phaC synthase of the strain directly affect the types of monomers that constitute PHA, the arrangement of monomers and the molecular weight of PHA, so as to obtain different physicochemical properties such as biodegradability, thermoplasticity, ductility and aeration. Material. These materials can be used to produce membranes, bags and bottles, heart valves, and more. Currently, there are more than 150 PHA structures inferred, but there are only 4 types of PHAs that have achieved large-scale commercial production. Therefore, obtaining novel phaC synthases from the environment is of great significance for the development of novel PHAs.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the problem of low separation efficiency of PHA-producing bacteria, and proposes to domesticate and screen strains in the manner of increasing the oil content and salt content of the medium from marine sediments, and use the oil-containing medium to obtain marine-derived polyhydroxyl Bacterial enrichment and separation method of fatty acid esters. Another object of the present invention is to provide a method that takes the marine sediments as the separation object and takes weeks as the time unit to gradually increase the salinity and oil content of the medium, enrich the bacteria that can metabolize oil in the bacterial community, and pass A method for enrichment and isolation of marine-derived polyhydroxyalkanoate-producing bacteria under extreme culture conditions to increase the likelihood of obtaining uncommon strains.

The first technical solution of the present invention is the configuration method of the marine-derived polyhydroxyalkanoate bacteria enrichment medium, the special feature of which is that the enrichment medium uses a basic medium with a gradient increase in salinity and oil content (minimal medium, MM medium), the configuration method of the medium, including the following steps:

(1) The enrichment medium is based on MM medium, adding oil and salt, and dissolving 1 mL of trace elements in 1 L of 0.1 mol/L HCl;

(2) Oil and sodium chloride (NaCl) are added in the enrichment medium according to the set ratio. The oil content of the MM medium starts from 1%, and the oil content and salt content are increased by 0.5 to 1% after each culture cycle. Starting from the salinity of Chen seawater from 35‰, after each culture cycle, by adding sodium chloride (NaCl), the salinity is increased to 10‰; the MM medium with a gradient increase ratio used in each culture cycle is named after the cycle. ;

(3) Adjust the pH value of the medium to 7.4, and autoclave for 20 min at 121 °C.

Preferably: the MM medium in step (1) consists of the following mass fraction wt%:

Na ₂ HPO ₄ 0.38 KH ₂ PO ₄ 0.265 (NH ₄ ) ₂ SO ₄ 0.05

MgSO ₄ 0.02 CuSO ₄ ·6H ₂ O 0.0155 ZnSO ₄ ·7H ₂ O 0.0153

CoCl ₂ ·6H ₂ O 0.022 MnCl ₂ ·4H ₂ O 0.00589 CaCl ₂ 0.78.

The second technical solution of the present invention is the inoculation method for the enrichment of the marine-derived polyhydroxyalkanoate bacteria, which is special in that it comprises the following steps:

(1) Mix marine sediments with MM1 medium at a ratio of 1:100, and shake them in a shaker for one cycle. The cultured sediment and medium mixture are called enriched culture medium; The enriched culture medium is inoculated into the next gradient MM2 medium at a ratio of 1:100, and so on, to complete 6-8 cycles of enrichment culture, and one cycle is 7-10 days;

(2) After 2 to 3 cycles of enrichment, carry out a separation to obtain a single colony with a single shape and color;

(3) Amplify the phaC synthase gene of the purified single colony by colony PCR, then separate the PCR product by agarose gel electrophoresis, judge whether the strain produces PHA according to whether there are obvious bands on the gel, and use PCR amplification 16S rRNA gene, first-generation sequencing to identify the taxonomic status of the strain.

As preferably: the described separation step of step (2) further comprises:

(2.1) Quickly shake the enriched culture medium for 30s to suspend the sediment, then take 30 μL of the suspension for gradient dilution, apply each dilution to the surface of the 2216E medium, and culture at 30°C for 2 to 7 days;

(2.2) According to the morphological characteristics of the colony, the colony is purified by the method of three-district streaking every day until a single colony with uniform color and shape is obtained.

As a preference: the isolated colonies in step (2) include: 11 strains of Firmicutes and 36 strains of Proteobacteria were obtained through their own separation, 21 of which contained the phaC synthase gene, and the potential PHA-producing strains isolated were as high as 45%; the obtained strains are from 11 genera, including: Acuticoccus, Alcanivorax, Bacillus, Halobacillus, Labrenzia ), Maritimibacter, Pelagibaca, Ponticaulis, Pseudooceanicola, Salipiger and Thalassospira.

The third technical solution of the present invention is the method for using polymerase chain reaction (PCR) to amplify the 16Sr RNA gene sequence to identify the taxonomic status of strains, which is special in that it includes the following steps:

(1) The primers are: 27F 5'-AGAGTTTGATCCTGGCTCAG-3', 1472R 5'-GGTTACCTTGTTACGACTT-3';

(2) The PCR reaction conditions were denaturation at 94°C for 6 min, followed by a reaction cycle of 45s at 94°C, 30s at 54°C, and 90s at 72°C for a total of 30 cycles, and finally extended at 72°C for 10min;

(3) Using the BLAST program of NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi), determine the type strain most similar to the sequence, and preliminarily judge the taxonomic status of the strain.

The fourth technical solution of the present invention is the method for identifying the potential ability of bacteria to synthesize PHA by using polymerase chain reaction (PCR) and agarose gel electrophoresis, which is special in that it includes the following steps:

(1) The primers are: phaC1F1 (5'-TGGARCTGATCCAGTAC-3') and phaC1R1 (5'-CGGGTTGAGRATGCTCTG-3');

(2) PCR reaction conditions are: denaturation at 94°C for 5 minutes, 1 minute at 94°C, 1 minute at 54°C, and 1 minute at 72°C as one reaction cycle, a total of 30 cycles of reaction, and finally extension at 72°C for 10 minutes;

(3) The PCR products were separated by agarose gel electrophoresis, using 1% agarose, and separated for 15 minutes at a voltage of 120V. It was confirmed that the strain had a clear band, indicating that the bacteria had the phaC synthase gene.

Compared with the prior art, the beneficial effects of the present invention:

(1) The enrichment culture method of marine PHA-producing bacteria according to the present invention is helpful to obtain a novel PHA-producing strain, thereby obtaining a novel phaC synthase gene.

(2) Using the method of the present invention to carry out the enrichment and separation of marine PHA-producing bacteria, compared with the method of separating non-marine environmental samples or directly separating without enrichment, a high proportion of PHA-producing bacteria coupled with oil metabolism can be obtained, and special It has important practical significance for promoting the development of different types of new PHA materials in the PHA industry.

Description of drawings

Figure 1 is a gel image of a PHA-producing strain screened by the present invention, the phaC synthase gene is amplified by polymerase chain reaction (PCR), and its DNA product is separated by agarose gel electrophoresis.

Detailed ways

The present invention will be described in further detail below in conjunction with embodiment:

Example

The marine sediment samples were taken from the sediments of Sanmenxia (E114°36'47.41", N22°28'35.33"), Dapeng Bay, Shenzhen. The sediment was cultured for 7 days in 99 mL of MM1 medium (salinity 35‰, oil content 1%), and 1 mL of suspension was taken with MM medium with salinity increased by 10‰ and oil content increased by 1%. Mix at 1:100. This cycle was repeated 6 times until the salinity of the medium increased to 95‰ and the oil content increased to 7%. Dilution coating was performed at the end of the third and sixth enrichment cycles.

The dilution ratios of the above-mentioned dilution coating were selected from -1, -2 and -3. A single colony was obtained by the method of three-zone streaking to obtain the pure bacteria of morphological color bacteria one. Colony PCR was performed using a single colony as a template to determine the taxonomic status and PHA-producing potential of the bacteria.

A method for identifying the taxonomic status of strains by amplifying the 16Sr RNA gene sequence by polymerase chain reaction (PCR), the primers are: 27F 5'-AGAGTTTGATCCTGGCTCAG-3', 1472R 5'-GGTTACCTTGTTACGACTT-3', denaturation at 94°C 6 min, followed by a reaction cycle of 45 s at 94 °C, 30 s at 54 °C, and 90 s at 72 °C, for a total of 30 cycles, and finally extended at 72 °C for 10 min. The PCR product was sent to a sequencing company (https://www.sangon.com/services_dnaseq.html), and the sequence was obtained by first-generation sequencing.

Then, use NCBI's BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the type strain most similar to the sequence, and preliminarily judge the taxonomic status of the strain.

Using polymerase chain reaction (PCR) and agarose gel electrophoresis to identify the potential ability of the strain to synthesize PHA, the primers are: phaC1F1 (5'-TGGARCTGATCCAGTAC-3') and phaC1R1 (5'-CGGGTTGAGRATGCTCTG-3'), The conditions were: denaturation at 94°C for 5 min, followed by a reaction cycle of 1 min at 94°C, 1 min at 54°C, and 1 min at 72°C, a total of 30 cycles, and finally extension at 72°C for 10 min. The PCR products were separated by agarose gel electrophoresis, using 1% agarose, and separated at 120V for 15 minutes. The gel image confirmed that there was a clear band at 500bp, which proved that the strain had the potential to produce PHA (as shown in Figure 1). .

Through 16S rRNA gene similarity identification, it was determined that the isolated bacteria belonged to 11 strains of Firmicutes and 36 strains of Proteobacteria, of which 21 strains contained phaC gene, and the potential PHA-producing strains isolated were as high as 45%. The obtained strains are from 11 genera, including: Acuticoccus, Alcanivorax, Bacillus, Halobacillus, Labrenzia, Maritimibacter genus Pelagibaca, Ponticaulis, Pseudooceanicola, Salipiger, and Thalassospira; the glue map identified a clear band at 500 bp, proving the strain Has the potential to produce PHA.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the claims of the present invention shall fall within the scope of the claims of the present invention.

Claims (7)

1. A preparation method of a marine polyhydroxy fatty acid ester-producing bacterium enrichment medium is characterized in that the enrichment medium uses a minimal medium (MM medium) with increased salinity and oil content gradient, and the preparation method of the medium comprises the following steps:

adding oil and salt into an enrichment medium based on an MM (media of cell culture), and dissolving 1mL of trace elements into 1L of 0.1 mol/LHCl;

adding oil and sodium chloride (NaCl) into the enrichment culture medium according to a set proportion, wherein the oil content of the MM culture medium is increased by 0.5-1% from 1%, the salt content is increased by 10% from 35% per mill of salinity of old seawater after each culture period; the gradient increasing proportion of MM medium used per cultivation cycle, named cycle;

adjusting the pH value of the culture medium to 7.4, and autoclaving at 121 ℃ for 20 min.

2. The method for configuring the marine polyhydroxyalkanoate-producing bacteria-enriched culture medium according to claim 1, wherein the MM culture medium comprises the following mass fraction wt%:

3. An inoculation method for enriching bacteria producing polyhydroxyalkanoate from a marine source is characterized by comprising the following steps:

mixing marine sediments with an MM1 culture medium in a ratio of 1:100, and fully shaking and culturing in a shaking table for one period, wherein a mixture of the cultured sediments and the culture medium is called an enrichment culture solution; inoculating an enrichment culture solution in the current MM1 culture medium into the next gradient MM2 culture medium in a ratio of 1:100, and so on, completing enrichment culture for 6-8 cycles, wherein the cycle is 7-10 days;

secondly, after enrichment is completed for 2-3 cycles, once separation is carried out to obtain single colonies with uniform shapes and colors;

amplifying the purified single colony phaC synthetase gene through colony PCR, separating PCR products by agarose gel electrophoresis, judging whether the bacterial strain produces PHA according to whether an obvious strip exists on the gel, amplifying the 16S rRNA gene by PCR, and identifying the classification status of the bacterial strain by first-generation sequencing.

4. The inoculation method for enriching the marine polyhydroxyalkanoate-producing bacteria according to claim 3, wherein the separation step further comprises:

(2.1) rapidly shaking the enrichment culture solution for 30s to suspend the sediment, then taking 30 mu L of suspension for gradient dilution, coating each dilution on the surface of a 2216E culture medium, and culturing for 2-7 days at the temperature of 30 ℃;

(2.2) purifying the colonies by using a three-zone streaking method according to the morphological characteristics of the colonies every day until single colonies with uniform color morphology are obtained.

5. The inoculation method for enriching the marine polyhydroxyalkanoate-producing bacteria according to claim 3, wherein the isolated colonies comprise: 11 strains of the firmicutes strain and 36 strains of the proteobacteria strain are obtained by the separation, wherein 21 strains contain phaC synthetase gene, and the separated potential PHA-producing strains are as high as 45 percent; the strains obtained were from 11 genera, including: the genera of the genera spinel (Acuticoccus), Alcanivorax (Alcanivorax), Bacillus (Bacillus), Halobacter (Halobacillus), Brenzeria (Labrenzia), Maritibacter, Phyllosphaera (Pelagibaca), Ponticulus, Pseudomonadaceae (Pseudonococeanicola), Salipiger and Thalassospira.

6. A method for identifying the classification status of strains by amplifying a 16Sr RNA gene sequence by utilizing a Polymerase Chain Reaction (PCR), which is characterized by comprising the following steps:

the first primer is: 27F 5'-AGAGTTTGATCCTGGCTCAG-3', 1472R 5'-GGTTACCTTGTTACGACTT-3';

The PCR reaction condition comprises firstly denaturation at 94 ℃ for 6min, then reaction at 94 ℃ for 45s, 54 ℃ for 30s and 72 ℃ for 90s for one cycle, 30 cycles in total, and finally 10min at 72 ℃;

thirdly, the model strain most similar to the sequence is determined by using the BLAST program (https:// blast.ncbi.nlm.nih.gov/blast.cgi) of NCBI, and the classification status of the strain is preliminarily judged.

7. A method for identifying potential PHA synthesis capability of a strain using Polymerase Chain Reaction (PCR) and agarose gel electrophoresis, comprising the steps of:

the first primer is: phaC1F1(5 '-TGGARCTGATCCAGTAC-3') and phaC1R1(5 '-CGGGTTGAGRATGCTCTG-3');

the PCR reaction conditions are as follows: denaturation at 94 deg.C for 5min, reacting at 94 deg.C for 1min, 54 deg.C for 1min, and 72 deg.C for 1min for 30 cycles, and extending at 72 deg.C for 10 min;

and thirdly, separating the PCR product by agarose gel electrophoresis, separating for 15min under the voltage of 120V by using 1% agarose, and determining that the strain has clear bands to characterize that the bacteria have the phaC synthetase gene.

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