Application of high-throughput screening method using droplet micro-fluidic chip in actinomycetes

A microfluidic chip and screening method technology, applied in the field of microbial biology, can solve the problem of high-throughput screening and detection without actinomycetes, and achieve the effect of wide detection and sorting time

Pending Publication Date: 2021-05-28
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, droplet microfluidics has been successfully applied in high-throughput screening of various bacteria and fungi, but there is no report on the application of droplet microfluidics to high-throughput screening and detection of actinomycetes.

Method used

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  • Application of high-throughput screening method using droplet micro-fluidic chip in actinomycetes
  • Application of high-throughput screening method using droplet micro-fluidic chip in actinomycetes
  • Application of high-throughput screening method using droplet micro-fluidic chip in actinomycetes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction of negative and positive strains with green fluorescent signal

[0040] On the basis of the sequence of the original enhanced green fluorescent protein gene egfp (as described in SEQ NO.1 in the sequence listing), ATG is used as the start codon, and all GTG codons in the expression frame are synonymously mutated to GTC or GTA to obtain the egfp (ATG) gene (as described in SEQ NO.2 in the sequence listing). On the basis of egfp(ATG), the initial ATG was replaced with ACG by point mutation method to obtain the egfp(ACG) gene (as described in SEQ NO.3 in the sequence listing). Select 5 heterologous promoters (Table 1): P gapdh(EL) , P rpsL(XC) , P rpsL(SG) , P rpsL(TP) , P erm*E , synthesize these promoter sequences, and use the synthetic sequences as templates to clone these promoters respectively; select 4 endogenous promoters at the same time (Table 1): P rpS12 , P gpmA , P pyk , P rpoA , and these promoters were cloned using the Streptomyc...

Embodiment 2

[0058] Embodiment two plate spore culture and collection

[0059] Take the spores (suspended in 20% glycerol solution) frozen at -80°C, dip a small amount of spore liquid with a sterile inoculation loop, and streak and passage on the R2YE solid plate. Plates were placed upside down in a 30°C incubator and cultured for 5-7 days until gray mature spores differentiated. When collecting, add 5 mL of sterile R2YE liquid medium that has passed through a 0.22 μm aqueous membrane to the surface of the plate, and repeatedly blow and suck the spores on the plate with a pipette to suspend them in the medium. Take 3 mL of the eluted spore suspension and add it to a sterile 8-layer lens paper filter device, and filter twice until the spores are in a monodisperse state in the medium (such as figure 2 shown). Take 20-30 μL of the filtered spore suspension and drop it on a hemocytometer, observe and count the spore concentration under a 20x objective lens. Dilute the spore suspension with...

Embodiment 3

[0060] Embodiment three droplet embedding method

[0061] Droplet embedding according to methods in published literature He, R., Ding, R., Heyman, J.A. et al. JInd Microbiol Biotechnol (2019) 46:1603. https: / / doi.org / 10.1007 / s10295- 019-02221-2: Wherein the water phase is 1mL and the spore concentration is 1×10 6 spore suspension per mL, add the above water phase into a 1mL syringe, the oil phase is 1mL oil containing stabilizer, also add into a 1mL syringe, and then the syringe with the water phase and the oil phase and the droplet form Microchip Unicom, try to adjust the ratio of the flow rate of the oil phase and the water phase to 1:1, 2:1 and 3:1, respectively, to generate droplets of different diameters ( Figure 3-B , C). When the ratio of spore concentration to the number of droplets is between 0.3-0.5 and the diameter of the obtained droplets is 80-100 μm, the formation ratio and detection speed of spore droplets are optimal. The size of the prepared droplet is rel...

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Abstract

The invention discloses an application of a high-throughput screening method using a droplet micro-fluidic chip in actinomycetes, actinomycete monospores can be embedded in a single droplet through the method, the single spores are stably formed in the droplet within the culture time of 0-7 days, the wide detection and sorting time is guaranteed, and in the aspect of detection, by using the method, functional promoters in actinomycetes can be detected at high throughput, or the strength of some unknown promoters can be identified, and promoter elements with proper strength can be screened for subsequent actinomycete strain modification; in the aspect of screening, the screening flux of the method can reach 105 strains per day, a mixed library of positive bacteria generating green fluorescence signals and negative bacteria not generating green fluorescence signals can be successfully sorted, and the enrichment rate of the sorted positive bacteria reaches 81.7% or above and is increased to 26 times compared with 3.1% before sorting.

Description

technical field [0001] The invention belongs to the field of microbial biotechnology. More specifically, the present invention relates to the application of a high-throughput screening method using a droplet microfluidic chip in actinomycetes. Background technique [0002] Actinobacteriaceae belongs to Actinomycetes, which is a kind of Gram-positive prokaryotes with high GC, hyphae growth and mainly asexual spore reproduction. The Actinomycetes include several genera such as Actinomycetes and Streptomyces, but there are great similarities between the genera in terms of living habits, morphological development and reproductive methods, for example: most are saprophytic; cultured on solid plates Most of them can differentiate into well-developed mycelium, and the mycelium is divided into basal mycelium responsible for absorbing nutrients and aerial mycelium extending to the surrounding space; aerial mycelium becomes sporocystus after maturity, and sporocystium further Differ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04G01N21/64
CPCC12Q1/045G01N21/6428
Inventor 涂然王猛张玥
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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