skip to primary navigationskip to content

Development of a microfluidic device for high-throughput analysis of genetic circuits in plant protoplasts, full application.

This project aims to develop a high-throughput screen for the analysis of promoter sequences driving expression of a reporter gene in plant protoplasts. We envisage this device to be applicable to a range of plant species and cell types, and by coupling it to libraries of regulatory elements identified by DNase-Seq, it will rapidly increase the rate of identifying promoters for biotechnological applications.

The Idea

DNA regulatory elements are fundamental parts for genetic circuit design. The generation and characterization of promoter libraries can greatly facilitate fine tuning of gene expression within a circuit. We have used DNase-Seq (Meyer and Liu 2014) to identify candidate regulatory elements (>50,000) controlling cell-preferential gene expression within maize leaves (Burgess and Reyna-Llorens; unpublished data). Current validation techniques involve fusing sequences to a reporter and a?nalysing expression in planta, which requires testing each element in an individual plant, either through transient biolistic transformation or the generation of stable transgenics (Brown et al. 2011). Applying these techniques to whole regulatory element libraries is not feasible at a laboratory scale.

This project aims to develop a high-throughput screen for the analysis of promoter sequences driving expression of a reporter gene in plant protoplasts. Protoplasts will be transformed by plasmid reporter constructs consisting of a regulatory region fused to a minimal promoter and a fluorescent protein. The expression level of fluorescent reporter within each protoplast will be questioned by laser in situ excitation in the sorting microfluidic device which will be also used to separate out individual protoplasts. Protoplasts will be sorted into two pools according to a user-specified threshold of fluorescence intensity (Abalde-Cela et al. 2015). In analysing a library of elements, this procedure will be performed iteratively at ever decreasing thresholds of fluorescence intensity to sort regulatory elements by their promoter activity. To determine which regulatory regions are present in each fraction, DNA will be extracted and sequenced using Illumina technology for each pool. We envisage this device to be applicable to a range of plant species and cell types, and by coupling it to libraries of regulatory elements identified by DNase-Seq, it will rapidly increase the rate of identifying promoters for biotechnological applications.

Who we are

Dr. Steven Burgess

Mr. Ivan Reyna-Llorens

Mr. Christian R. Boehm

Dr. Sara Abalde-Cela

Dr. Paul Bennett

Implementation

Implementation Stage 1 - Establishment of procedures for protoplast isolation and handling, involving:

  • fabrication of a PDMS/glass microfluidic device designed for analysis of plant protoplasts by conventional soft lithography methods used in the Abell lab (Abalde-Cela et al. 2015).

  • establishment of protoplast isolation from Marchantia (according to Ono et al. 1979, modified) and maize leaves (according to Sheen 2002). Protoplast cells are more fragile than other cells since they do not possess a cell wall. This could have an effect on the ability of protoplasts to withstand the forces generated within a microfluidic device, although analysis of protoplasts using microfluidics has previously been reported (Ko et al. 2006). If protoplasts are found to be too fragile, we will switch to using algal or cyanobacterial cells, for which procedures have already been established and a suitable device created (Abalde-Cela et al. 2015).

  • establishment of a robust procedure for protoplast transformation (e.g. based on commonly used methods such as PEG and electroporation) (Ono et al. 1979; Sheen et al. 2001) building on experience from Haseloff lab.

  • test of protoplast stability in the microfluidic device.

Stage 2 - Development of tools for quantification of a fluorescent reporter in protoplasts, including:

  • a reporter construct created using Golden Gate Cloning, which is routinely employed in the Hibberd Lab. All parts will be compatible with the Plant MoClo system (Engler et al. 2014), and made publically available via Addgene. Vectors will embrace a site for insertion of gene regulatory regions of interest, which will drive a codon optimized mVenus, together with mTurquoise2 constitutively expressed under the control of the UbiQ promoter. In order to test the system, we will clone in the PEPC promoter of maize to drive the expression of various reporter genes. All the promoter level 0 Golden Gate modules are already available in the Hibberd lab.

  • a multi-part optical set-up for fluorescence detection in order to quantify the level of reporter expression, as previously used in the Abell lab.

  • an open source software tool used for analysis of generated data generated and sorting of protoplasts according a user-specified fluorescence threshold. We have access to data generated by a similar device. This data will be used to develop the analysis tool while our device is under construction.

Time line (please contact sjb287@cam.ac.uk for spreadsheet if necessary):

Month 1

1.1-Microfluidic device construction

2.1-Software development

3.1-Protoplast isolation of wild type and transformed lines

4.1- Plasmid construction

Month 2

2.1-Software development

3.1-Protoplast isolation of wild type and transformed lines

4.1- Plasmid construction

3.2-Protoplast transformation (Depending on 3.1 and 4.1)

5.1-Test chlorophyll content and protoplast integrity (Depending on 1.1, 2.1, 3.1)

Month 3

2.1-Software development

3.3-Ongoing generation of protoplasts

5.1-Test chlorophyll content and protoplast integrity (Depending on 1.1, 2.1, 3.1)

5.2 Protoplast sorting by promoter activity (Depending on 3.2, 4.1 and 5.1)

Month 4

2.1-Software development

3.3-Ongoing generation of protoplasts

5.2 Protoplast sorting by promoter activity (Depending on 3.2, 4.1 and 5.1

Month 5

6.1 Write up (Depending on 5.2)

3.3-Ongoing generation of protoplasts

5.2 Protoplast sorting by promoter activity (Depending on 3.2, 4.1 and 5.1

Month 6

6.1 Write up.

Benefits and outcomes

(1) An open source golden-gate reporter system for analysis of maize regulatory elements.

(2) A microfluidic device for analysis of plant protoplasts. Our approach allows the use of individual cells rather than entire organisms to test the effect of regulatory regions on reporter activity, facilitating screening of large libraries of genetic elements.

(3) Open-source software for microfluidic analysis. Current methods rely on commercial software such as LabView (http://www.ni.com/labview/) or MatLab, can be difficult to use for non-experts, and are not open access. We will design accessible software tools to benefit the wider community.

(4) A blog with regular updates about the progress of the project to inform the community and conduct our work in an open manner.

(5) Publication of our method and results firstly in bioRxiv then in an open access journal depending on outcomes.

Budget

(1) Fabrication of a Microfluidic device HR Photomask, £90

Photopolymer, £100

Photopolymer developer (PGMEA), £20

Silicon wafers, £30

Sylgard PDMS - 0.5 Kg of polymer + curing agent kit, £50

Aquapel, 5mL, £20 T

ubing, 3 metres - for flow pumping, £100

Syringes and needles, £50

Surfactant for droplet formation, free

Subtotal, £460

(2)

Generation of reporter construct Promoters modules for Golden gate cloning, Free Codon optimized reporter gene synthesis (~1500 bp), £270.0

Restriction Enzymes (BsaI and BpiI), £70.0

T4 DNA ligase, £45.0

ATP, £30.0

QIAprep Spin Miniprep Kit (50), £50.0

DNeasy Plant Mini Kit (50), £150.0

Sanger sequencing service (~20 rxns), £100.00

SYBR Green Taq ready mix, £120.00

Oligonucleotides, £100.00

Subtotal, £935.00 Generation of protoplasts & protoplast transformation Driselase (5g, Sigma), £175.07 Hemicellulase (300KU, Sigma), £81.14

Subtotal, £256.21

Growing Space

A shelf at the PGF - £14.34 per day for 150 days, £2151

Final Total: £3802.21

Contingency budget

Extra sanger sequencing (~20 rxns), £100.00

Gene synthesis of more reporter constructs, £270.00

About

The Synthetic Biology Strategic Research Initiative provides a hub for anyone interested in Synthetic Biology at the University of Cambridge, including researchers, commercial partners and external collaborators. 

Subscribe to our mailing list