DNA-BOT: a low-cost, automated DNA assembly platform for synthetic biology

Storch, Marko; Haines, Matthew; Baldwin, Geoff

doi:10.1093/synbio/ysaa010

Cited by 57 publications

(45 citation statements)

References 30 publications

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“…Importantly, this implementation was carried out simply by assembling regulators and targets using BASIC DNA assembly without re-optimisation of the regulator for different target genes. This extensibility is essential for the simultaneous regulation of multiple genes in metabolic engineering, layered genetic control, and advanced genetic circuit applications, while scalability is possible through our recently demonstrated low cost automation platform for BASIC assembly (26).…”

Section: Discussionmentioning

confidence: 99%

“…Here, we set out to design a universal, modular framework for the facile implementation of trans -acting sRNA-based control using verified, re-usable target sequences, namely m odular A rtificial R NA i nterference (mARi). BASIC DNA assembly provides a modular, standardized and automatable (26) framework for genetic structure based on orthogonal, computationally designed linkers (27). We exploited this by targeting the DNA linkers used in the construction of gene expression cassettes and operons.…”

Section: Introductionmentioning

confidence: 99%

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A modular RNA interference system for multiplexed gene regulation

Dwijayanti

Storch

Stan

et al. 2019

Preprint

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17The behaviour of engineered biological systems can be subject to deviation from design due to 18 unpredictable changes in growth conditions, host response and metabolic load. Control engineering 19 approaches are key to automatically maintain robust performance of designed systems despite such 20 variations. RNA-controlled gene expression creates new possibilities for systematic, fast-responding 21 and resource-efficient implementations of synthetic biology systems in living cells. Here, we 22 demonstrate that a rational, modular design framework can provide an extensible platform for 23 modular Artificial RNA interference (mARi) for robust, portable and multiplexed post-transcriptional 24 regulation in Escherichia coli. We used mARi to construct a host-aware negative feedback control 25 system that regulated protein production in response to cellular stress. We demonstrated that this 26 mARi-based feedback control system resulted in an improvement in protein production up to 5-fold 27 and was robust to perturbations in temperature, medium, scale, cell strain and gene product without 28 further tuning. We anticipate that the use of robust RNA-based genetic control within an extensible 29 DNA assembly framework, such as mARi, will have widespread applications in the realisation of 30 sophisticated engineered biological systems, where dynamic regulation can provide improvements in 31 gene circuit stability and microbial production. 32 33 Main 34 Cells use complex regulatory networks to appropriately sense, respond, and adapt to changes 35 in their internal states and environment. One widely employed mechanism for adaptation relies on 36 post-transcriptional regulation via small RNAs (sRNAs) 1,2 . sRNAs facilitate rapid, reversible, dynamic, 37 and efficient signal propagation and typically act to coordinate and synchronize multiple signals 38 through sequence-specific and transient RNA-RNA interactions 2,3 , frequently leading to down-39 regulation of target gene expression 4 . In bacterial systems, trans-acting sRNAs rather than cis-acting 40 sRNAs are reported to be especially effective in silencing gene expression due to their longer half-life 5 . 41 2The features of natural trans-encoded sRNAs have inspired versatile genetically encoded 42 controllers for metabolic engineering and synthetic biology purposes [6][7][8][9][10][11] . sRNAs have a modular 43 structure that lends itself to targeting of different genes. Specifically, the seed sequence that targets 44 an mRNA can be altered to bind new targets, while retaining the native sRNA scaffold [12][13][14] (Figs. 1a and 45 2a). The scaffold sequence is responsible for recognition by RNA chaperones, such as the host factor-46 1 (Hfq) protein. These abundant helper proteins are highly conserved in a range of organisms 15-18 as 47 part of natural cellular regulation 19,20 . Use of endogenous regulatory machinery makes synthetic 48 sRNA-based regulation portable, fast and lean when compared to other regulators such as 49 transcription factors, zinc fingers...

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A modular RNA interference system for multiplexed gene regulation

Dwijayanti

Storch

Stan

et al. 2019

Preprint

Self Cite

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“…To achieve this, tools and techniques typically used for biological applications were considered, therefore bridging the gap between biology and chemistry. Specifically, liquid handlers (an acoustic liquid handler [29][30][31][32] and the Opentrons [33][34][35][36] ) and a microplate reader were utilised. The acoustic liquid handler dispenses nanodroplet (as small as 2.5 nL droplets) of a solution into a 96-well plate.…”

Section: Introductionmentioning

confidence: 99%

“…[43][44][45][46] Recently, an acoustic liquid handler was used for the preparation of boronic acid libraries. [47] The Opentrons is a liquid handler typically used for biological applications such as polymer chain reaction (PCR), [33,34] nucleic acid extraction and purification, DNA assembly, [35] protein preparation [36] as well as fungal sample preparation. [48] It can also be used for making dilutions.…”

Section: Introductionmentioning

confidence: 99%

Robotic Catalysis: A High‐Throughput Method for Miniature Screening of Mesoporous Metal Oxides**

et al. 2020

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We have developed a high-throughput system to synthesise and explore up to 96 heterogeneous catalysts at the same time.The system was developed as a proof of concept, using a standard glass plate and a 3D printed 96-well plate. Nanodroplets of catalyst formulations were transferred to the glass plate using an acoustic liquid handler and upon heat treatments, the miniature mesoporous metal oxide (MMO) catalysts were formed. The 3D printed bottomless 96-well plate was fixed to the glass plate, to give 96 individual wells, each containing a catalyst. Four catalyst plates were prepared (Co 3 O 4 -, Au/Co 3 O 4 -, Pd/Co 3 O 4 -and Co/Mn-MMO) to be screened for their activity in the oxidation of morin, as a model reaction. The observed reaction rates (k obs ) for each catalyst were calculated to identify the most active catalyst. The general method described herein requires microscopic amounts of catalysts with derivates of the catalyst's composition.

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“…Many of the widely adopted DNA assembly standards (e.g., BASIC [2], MoClo [6] or Start-Stop assembly [7]) are "one-pot", i.e. involve mixing all of a construct's DNA parts together before ligating them in a single reaction.…”

mentioning

confidence: 99%

A linear programming-based strategy to save pipette tips in automated DNA assembly

Sechkar

Tuza

Stan

2021

Preprint

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Laboratory automation and mathematical optimisation are key to improving the efficiency of synthetic biology research. While there are algorithms optimising the construct designs and synthesis strategies for DNA assembly, the optimisation of how DNA assembly reaction mixes are prepared remains largely unexplored. Here, we focus on reducing the pipette tip consumption of a liquid-handling robot as it delivers DNA parts across a multi-well plate where several constructs are being assembled in parallel. We propose a linear programming formulation of this problem based on the capacitated vehicle routing problem, along with an algorithm which applies a linear programming solver to our formulation, hence providing a strategy to prepare a given set of DNA assembly mixes using fewer pipette tips. The algorithm performed well in randomly generated and real-life scenarios concerning several modular DNA assembly standards, proving capable of reducing the pipette tip consumption by up to 61% in large-scale cases. Combining automatic process optimisation and robotic liquid-handling, our strategy promises to greatly improve the efficiency of DNA assembly, either used alone or in combination with other algorithmic methods.

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DNA-BOT: a low-cost, automated DNA assembly platform for synthetic biology

Cited by 57 publications

References 30 publications

A modular RNA interference system for multiplexed gene regulation

A modular RNA interference system for multiplexed gene regulation

Robotic Catalysis: A High‐Throughput Method for Miniature Screening of Mesoporous Metal Oxides**

A linear programming-based strategy to save pipette tips in automated DNA assembly

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