Discrimination method for mutation-induced unicellular organism and microfluidic device used therefor

Patent metadata

Field	Value
Publication number	US-9650659-B2
Application number	US-201414760607-A
Country	US
Kind code	B2
Filing date	Jan 14, 2014
Priority date	Jan 21, 2013
Publication date	May 16, 2017
Grant date	May 16, 2017

Abstract

The present invention discloses a microfluidic photoreaction system and a method for screening a single cell organism having changed light-response characteristics. According to the present invention, an improved single cell organism can be effectively screened based on phototaxis using a microfluidic system. Specifically, easy monitoring at the cellular level is possible, and a mutant strain having an increased response and/or sensitivity to light can be easily and rapidly screened by various analyses, including statistical analysis of collected results. Thus, the present invention can be effectively used to investigate the correlation between phototaxis and photoconversion efficiency and to screen a single cell organism having increased photosynthetic efficiency.

First claim

The invention claimed is:

1. A method of screening to identify a single cell organism having a mutation that affects its phototaxis, the method comprising the steps of:
(a) obtaining a collection of single cell organisms, wherein one or more of the single cell organisms may have a mutation that affects its phototaxis;
(b) pretreating the collection of single cell organisms by culturing each organism under continuous light, wherein the light has a wavelength of 540-600 nm or 430-500 nm and an intensity of 20-50 μmol of photons m −2 s −1 , and then culturing each organism in the dark;
(c) irradiating the collection of single cell organisms with light to induce phototaxis;
(d) calculating the phototactic indices of each culture of the single cell organisms; and
(e) selecting a single cell organism as the desired single cell organism having a mutation in phototactic ability, if the phototactic indices of the selected single cell organism differ from those of a control group of phototactically normal single cell organisms.

2. The method of claim 1 , wherein the single cell organism is cultured to the exponential phase of the cell growth cycle in the pretreatment step.

3. The method of claim 1 , wherein the single cell organism is derived from either single colonies or multiple colonies.

4. The method of claim 1 , wherein the phototaxis is either positive phototaxis or negative phototaxis.

5. The method of claim 4 , wherein the phototaxis is negative phototaxis that is induced by irradiating light having a wavelength of 540-600 nm or 430-500 nm and an intensity of 20-50 μmol of photons m −2 S −1 .

6. The method of claim 1 , wherein the phototactic indices of the single cell organisms are calculated through the measurement of at least one of the light response and the light sensitivity.

7. The method of claim 1 , wherein the desired single cell organism having the mutation has one or more improved indices of photosynthesis, including changes in photosynthetic mechanisms including photosynthetic pigments, photosynthesis efficiency and photoconversion efficiency, and growth rate in comparison with the control group.

8. The method of claim 1 , wherein the phototactic indices include: (i) the ratio of the number of single cell organisms that moved per unit time in response to light to the total number of single cell organisms used; (ii) histogram peak analysis based on the distribution of the number of single cell organisms that moved per unit time; or (iii) the average time taken for single cell organisms to move a unit distance, the speed of the movement, or the variation in their position.

9. The method of claim 1 , further comprising measuring the phototactic indices for the screened single cell organisms.

10. The method of claim 9 , wherein the phototactic indices include changes in photosynthetic mechanisms including photosynthetic pigments, photosynthesis efficiency and photoconversion efficiency.

11. The method of claim 1 , wherein the single cell organisms are microalgae.

12. The method of claim 11 , wherein the microalgae are green algae, diatoms, red algae, flagellates, light green algae, brown flagellates, yellow-green algae, dinoflagellates, or blue-green algae.

13. The method of claim 12 , wherein the Green algae are Chlamydomonas spp.

14. The method of claim 13 , wherein the Chlamydomonas sp. is Chlamydomonas reinhardtii.

Assignees

• Univ Korea Res & Bus Found

Inventors

• Sim Sang Jun
• Kim Young Hwan
• Kwak Ho Seok

Classifications

• G01N2035/00158

  Elements containing microarrays, i.e. "biochip" · CPC title

• B01L3/5027

  by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip · CPC title

• C12Q1/04 Primary

  Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)} · CPC title

• C12Q1/02 Primary

  involving viable microorganisms · CPC title

• G01N35/08

  using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis · CPC title