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Smart DNA Fabrication Using Sound Waves: Applying Acoustic Dispensing Technologies to Synthetic Biology.

Kanigowska P, Shen Y, Zheng Y, Rosser S, Cai Y - J Lab Autom (2015)

Bottom Line: This noncontact, tipless, low-volume dispensing technology minimizes the possibility of cross-contamination and potentially reduces the costs of reagents and consumables.We were able to successfully downscale PCRs and the popular one-pot DNA assembly methods, Golden Gate and Gibson assemblies, from the microliter to the nanoliter scale with high assembly efficiency, which effectively cut the reagent cost by 20- to 100-fold.We envision that acoustic dispensing will become an instrumental technology in synthetic biology, in particular in the era of DNA foundries.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Edinburgh, The King's Buildings, Edinburgh, UK.

No MeSH data available.


Gibson assembly reaction setup by Echo. (A) The pPC025plasmid was split into two overlapping fragments at the middle ofthe ampicillin resistance gene and the RFP ORF. Two fragments weregenerated with 40 bp overlap at both ends and then assembled by theGibson assembly reaction. (B) Gel electrophoresisconfirming the successful PCR amplification of both fragments.(C) Successful Gibson assembly product gives riseto red bacterial colonies. The assembly efficiency was so high thatno background colonies (white) were observed. Negative controlreactions, which had only one fragment in the reactions, yielded nocolonies. (D) Sequencing verification of both assemblyjunctions shows 100% assembly accuracy. (E)Cost-effectiveness and assembly efficiency comparison of differentreaction volumes for Gibson assembly.
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fig2-2211068215593754: Gibson assembly reaction setup by Echo. (A) The pPC025plasmid was split into two overlapping fragments at the middle ofthe ampicillin resistance gene and the RFP ORF. Two fragments weregenerated with 40 bp overlap at both ends and then assembled by theGibson assembly reaction. (B) Gel electrophoresisconfirming the successful PCR amplification of both fragments.(C) Successful Gibson assembly product gives riseto red bacterial colonies. The assembly efficiency was so high thatno background colonies (white) were observed. Negative controlreactions, which had only one fragment in the reactions, yielded nocolonies. (D) Sequencing verification of both assemblyjunctions shows 100% assembly accuracy. (E)Cost-effectiveness and assembly efficiency comparison of differentreaction volumes for Gibson assembly.

Mentions: The HcKan_P plasmid (2.8 kb, diluted to 10 ng/µl) was used as the acceptorvector. This plasmid carries a KanR selectable marker, along with a RFPcassette flanked by a pair of outward-facing BsaI sites. We amplified thepromoter pMBP1 (500 bp) directly from yeast BY4741 (MATa,leu2∆0 met15∆0 ura3∆0 his3∆1) genomic DNA with primersYCp2395 and YCp2396 and added a pair of inward-facing BsaI sites to flankthe promoter part (Fig. 2A). The PCR product was purified using aPureLink PCR purification kit (Life Technologies) and diluted to 20 ng/µl.The 4 bp overhangs were designed in such a way that the promoter can beefficiently assembled into the acceptor vector. Bacteria carrying theresidual RFP plasmid will give a bright red pigment, which would facilitatethe visual identification of correct assembled clones (white colonies; seeFig.2C).


Smart DNA Fabrication Using Sound Waves: Applying Acoustic Dispensing Technologies to Synthetic Biology.

Kanigowska P, Shen Y, Zheng Y, Rosser S, Cai Y - J Lab Autom (2015)

Gibson assembly reaction setup by Echo. (A) The pPC025plasmid was split into two overlapping fragments at the middle ofthe ampicillin resistance gene and the RFP ORF. Two fragments weregenerated with 40 bp overlap at both ends and then assembled by theGibson assembly reaction. (B) Gel electrophoresisconfirming the successful PCR amplification of both fragments.(C) Successful Gibson assembly product gives riseto red bacterial colonies. The assembly efficiency was so high thatno background colonies (white) were observed. Negative controlreactions, which had only one fragment in the reactions, yielded nocolonies. (D) Sequencing verification of both assemblyjunctions shows 100% assembly accuracy. (E)Cost-effectiveness and assembly efficiency comparison of differentreaction volumes for Gibson assembly.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2 - License 3
Show All Figures
getmorefigures.php?uid=PMC4814025&req=5

fig2-2211068215593754: Gibson assembly reaction setup by Echo. (A) The pPC025plasmid was split into two overlapping fragments at the middle ofthe ampicillin resistance gene and the RFP ORF. Two fragments weregenerated with 40 bp overlap at both ends and then assembled by theGibson assembly reaction. (B) Gel electrophoresisconfirming the successful PCR amplification of both fragments.(C) Successful Gibson assembly product gives riseto red bacterial colonies. The assembly efficiency was so high thatno background colonies (white) were observed. Negative controlreactions, which had only one fragment in the reactions, yielded nocolonies. (D) Sequencing verification of both assemblyjunctions shows 100% assembly accuracy. (E)Cost-effectiveness and assembly efficiency comparison of differentreaction volumes for Gibson assembly.
Mentions: The HcKan_P plasmid (2.8 kb, diluted to 10 ng/µl) was used as the acceptorvector. This plasmid carries a KanR selectable marker, along with a RFPcassette flanked by a pair of outward-facing BsaI sites. We amplified thepromoter pMBP1 (500 bp) directly from yeast BY4741 (MATa,leu2∆0 met15∆0 ura3∆0 his3∆1) genomic DNA with primersYCp2395 and YCp2396 and added a pair of inward-facing BsaI sites to flankthe promoter part (Fig. 2A). The PCR product was purified using aPureLink PCR purification kit (Life Technologies) and diluted to 20 ng/µl.The 4 bp overhangs were designed in such a way that the promoter can beefficiently assembled into the acceptor vector. Bacteria carrying theresidual RFP plasmid will give a bright red pigment, which would facilitatethe visual identification of correct assembled clones (white colonies; seeFig.2C).

Bottom Line: This noncontact, tipless, low-volume dispensing technology minimizes the possibility of cross-contamination and potentially reduces the costs of reagents and consumables.We were able to successfully downscale PCRs and the popular one-pot DNA assembly methods, Golden Gate and Gibson assemblies, from the microliter to the nanoliter scale with high assembly efficiency, which effectively cut the reagent cost by 20- to 100-fold.We envision that acoustic dispensing will become an instrumental technology in synthetic biology, in particular in the era of DNA foundries.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Edinburgh, The King's Buildings, Edinburgh, UK.

No MeSH data available.