US 11,851,651 B2
Automated methods for scalable, parallelized enzymatic biopolymer synthesis and modification using microfluidic devices
James Banal, Cambridge, MA (US); Joseph Don Berleant, Cambridge, MA (US); Tyson Shepherd, Arlington, MA (US); and Mark Bathe, Cambridge, MA (US)
Assigned to MASSACHUSETTS INSTITUTE OF TECHNOLOGY, Cambridge, MA (US)
Filed by Massachusetts Institute of Technology, Cambridge, MA (US)
Filed on Jun. 19, 2018, as Appl. No. 16/012,583.
Claims priority of provisional application 62/521,612, filed on Jun. 19, 2017.
Prior Publication US 2018/0362969 A1, Dec. 20, 2018
Int. Cl. B01L 9/00 (2006.01); C12N 15/10 (2006.01); G16B 35/00 (2019.01); G16C 20/60 (2019.01); C12P 19/34 (2006.01); B01J 19/00 (2006.01); B01L 3/00 (2006.01); C12Q 1/6811 (2018.01); G16B 40/00 (2019.01)
CPC C12N 15/1068 (2013.01) [B01J 19/0046 (2013.01); C12P 19/34 (2013.01); G16B 35/00 (2019.02); G16C 20/60 (2019.02); B01J 2219/0065 (2013.01); B01J 2219/00675 (2013.01); B01J 2219/00722 (2013.01); B01J 2219/00725 (2013.01); B01J 2219/00729 (2013.01); B01L 3/502784 (2013.01); B01L 3/502792 (2013.01); B01L 2400/0427 (2013.01); C12N 15/1093 (2013.01); C12Q 1/6811 (2013.01); C12Q 2565/629 (2013.01); G16B 40/00 (2019.02)] 43 Claims
 
1. A method for synthesizing a biopolymer having a user-defined sequence of component building blocks on a microfluidic system, the method comprising:
(a) forming, on a microfluidic device, first, second, and third droplets, wherein the microfluidic device comprises:
(i) a multiplicity of spatially distinct addressed locations;
(ii) a multiplicity of distinct fluid reservoirs; and
(iii) a microfluidic handling system that can actuate (1) splitting of droplets from each of the multiplicity of reservoirs through motive force induced by electric potential or optical excitation at the fluid reservoirs, and (2) movement of droplets from each of the addressed locations to another of the addressed locations through motive force induced by electric potential or optical excitation at the addressed locations,
wherein each of the multiplicity of fluid reservoirs is located at a spatially distinct addressed location on the microfluidic device,
wherein a first of the multiplicity of fluid reservoirs comprises a solution comprising a component initiation sequence,
wherein a second of the multiplicity of fluid reservoirs comprises a solution comprising a first component building block,
wherein a third of the multiplicity of fluid reservoirs comprises a solution comprising an attachment catalyst that can catalyze the attachment of the component building block to the component initiation sequence and/or a further component building block, and
optionally wherein a fourth or further of the multiplicity of fluid reservoirs comprises a solution comprising a further component building block;
wherein the first droplet comprises a component initiation sequence,
wherein the first droplet is formed by splitting from the first reservoir,
wherein the second droplet comprises a first component building block,
wherein the second droplet is formed by splitting from the second reservoir,
wherein the third droplet comprises an attachment catalyst,
wherein the third droplet is formed by splitting from the third fluid reservoir,
wherein the splitting comprises moving a droplet of fluid away from a fluid reservoir, and
wherein each of the droplets is formed at a distinct addressed location on the microfluidic device;
(b) combining the first, second, and third droplets to form a combined droplet,
wherein the combining comprises moving each of the droplets to the same addressed location on the microfluidic device,
wherein the combining is performed under conditions suitable for the attachment catalyst to attach the component initiation sequence to the first component building block within the combined droplet;
(c) forming, on the microfluidic device, a further droplet comprising a component building block,
wherein forming the further droplet comprises splitting a droplet from the second, or fourth or further reservoir, and
optionally forming an additional droplet comprising an attachment catalyst,
wherein forming the additional droplet comprises splitting a droplet from the third reservoir;
(d) combining the combined droplet with the further droplet and, optionally, with the additional droplet, to synthesize a biopolymer,
wherein the combining comprises moving the further droplet and/or the combined droplet and, optionally, the additional droplet, to the same addressed location, and
wherein the combining is performed under conditions suitable for the attachment catalyst to attach the component building block from the further droplet to the component building block within the combined droplet;
(e) optionally repeating steps (c) and (d) one or more times to perform step wise addition of component building blocks to the biopolymer; and
(f) manipulating, purifying, or isolating the synthesized biopolymer on the microfluidic device,
wherein the sequence of the synthesized biopolymer is defined by the order in which droplets comprising component building blocks are combined with the combined droplet, and
wherein the order in which the droplets are combined is controlled by the user.
 
42. A method for synthesizing a biopolymer having a user-defined sequence of component building blocks on a microfluidic system, the method comprising:
(a) forming, on a microfluidic device, a first and second droplets, wherein the microfluidic device comprises:
(i) a multiplicity of spatially distinct addressed locations;
(ii) a multiplicity of distinct fluid reservoirs; and
(iii) a microfluidic handling system that can actuate (1) splitting of droplets from each of the multiplicity of reservoirs through motive force induced by electric potential or optical excitation at the fluid reservoirs, and (2) movement of droplets from each of the addressed locations to another of the addressed locations through motive force induced by electric potential or optical excitation at the addressed locations,
wherein each of the multiplicity of fluid reservoirs is located at a spatially distinct addressed location on the microfluidic device,
wherein a first of the multiplicity of fluid reservoirs comprises a solution comprising a component initiation sequence,
wherein a second of the multiplicity of fluid reservoirs comprises
(i) a component building block; and
(ii) an attachment catalyst that can catalyze the attachment of the component building block to the component initiation sequence and/or another component building block;
optionally wherein a third or further of the multiplicity of fluid reservoirs comprises
(i) a solution comprising a component building block; and
(ii) an attachment catalyst that can catalyze the attachment of the component building block to the component initiation sequence and/or another component building block;
wherein the first droplet comprises a component initiation sequence and is formed by splitting from the first of the multiplicity of fluid reservoirs,
wherein the second droplet comprises a component building block and an attachment catalyst and is formed by splitting from the second of the multiplicity of fluid reservoirs,
wherein the splitting comprises moving a droplet of fluid away from a fluid reservoir, and
wherein each of the droplets is formed at a distinct addressed location on the microfluidic device;
(b) combining the first and second droplets to form a combined droplet,
wherein the combining comprises moving each of the droplets to the same addressed location on the microfluidic device,
wherein the combining is performed under conditions suitable for the attachment catalyst to attach the component initiation sequence to the component building block within the combined droplet;
(c) forming, on the microfluidic device, a third or further droplet comprising a component building block and attachment catalyst,
wherein forming the further droplet comprises splitting a droplet from the second, third, or further of the multiplicity of fluid reservoirs;
(d) combining the combined droplet with the third or further droplet to synthesize a biopolymer,
wherein the combining comprises moving the further droplet and/or the combined droplet to the same addressed location, and
wherein the combining is performed under conditions suitable for the attachment catalyst to attach the component building block from the third droplet to the component building block within the combined droplet;
(e) optionally repeating steps (c) and (d) one or more times to perform step wise addition of component building blocks to the biopolymer; and
(f) manipulating, purifying, or isolating the synthesized biopolymer on the microfluidic device,
wherein the sequence of the synthesized biopolymer is defined by the order in which droplets comprising component building blocks are combined with the combined droplet, and
wherein the order in which the droplets are combined is controlled by the user.