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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.


Golden Gate assembly setup by Echo. (A) A promoter pMBP1 wasamplified from the yeast genome to add appropriate Golden Gate sequences(BsaI recognition sites + 4 bp overhangs). The acceptor vector HcKan_Pplasmid carries a RFP cassette, which is flanked by corresponding GoldenGate sequences to uptake the pMBP1 part in the Golden Gate reaction.(B) Gel electrophoresis indicates successful amplificationof pMBP1. (C) Left: Successful assembled DNA gives rise towhite colonies, while the residual acceptor vector yields red colonies.Right: Negative control, which contained only the acceptor vector in theGolden Gate reaction, yielded only red colonies. (D) Sequencingverification of both assembly junctions shows 100% assembly accuracy.(E) Cost-effectiveness and assembly efficiency comparisonof different reaction volumes for Golden Gate assembly.
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fig3-2211068215593754: Golden Gate assembly setup by Echo. (A) A promoter pMBP1 wasamplified from the yeast genome to add appropriate Golden Gate sequences(BsaI recognition sites + 4 bp overhangs). The acceptor vector HcKan_Pplasmid carries a RFP cassette, which is flanked by corresponding GoldenGate sequences to uptake the pMBP1 part in the Golden Gate reaction.(B) Gel electrophoresis indicates successful amplificationof pMBP1. (C) Left: Successful assembled DNA gives rise towhite colonies, while the residual acceptor vector yields red colonies.Right: Negative control, which contained only the acceptor vector in theGolden Gate reaction, yielded only red colonies. (D) Sequencingverification of both assembly junctions shows 100% assembly accuracy.(E) Cost-effectiveness and assembly efficiency comparisonof different reaction volumes for Golden Gate assembly.

Mentions: With Golden Gate assembly, we successfully assembled DNA at a 50 nL reaction volume(typically 15 µL reactions when performed manually), and at the 250 and 500 nLscales the assembly efficiencies are higher than those of the manual control. Thisleads to at least a 30-fold reduction in reagent use when performing Golden Gatereactions using Echo. We did observe vector background in the assembly (redcolonies, as shown in Fig.3C). There are several ways we can overcome this problem.First, instead of using RFP for screening, we can use the toxic ccdB gene, whichcannot give rise to background colonies in a nonpermissive transformation host.Second, we can add a higher concentration of the BsaI enzyme in the Golden Gatemaster mix to further digest the residual acceptor vector. Finally, we may be ableto reduce the background by extending the BsaI digestion step in the incubation.


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)

Golden Gate assembly setup by Echo. (A) A promoter pMBP1 wasamplified from the yeast genome to add appropriate Golden Gate sequences(BsaI recognition sites + 4 bp overhangs). The acceptor vector HcKan_Pplasmid carries a RFP cassette, which is flanked by corresponding GoldenGate sequences to uptake the pMBP1 part in the Golden Gate reaction.(B) Gel electrophoresis indicates successful amplificationof pMBP1. (C) Left: Successful assembled DNA gives rise towhite colonies, while the residual acceptor vector yields red colonies.Right: Negative control, which contained only the acceptor vector in theGolden Gate reaction, yielded only red colonies. (D) Sequencingverification of both assembly junctions shows 100% assembly accuracy.(E) Cost-effectiveness and assembly efficiency comparisonof different reaction volumes for Golden Gate assembly.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3-2211068215593754: Golden Gate assembly setup by Echo. (A) A promoter pMBP1 wasamplified from the yeast genome to add appropriate Golden Gate sequences(BsaI recognition sites + 4 bp overhangs). The acceptor vector HcKan_Pplasmid carries a RFP cassette, which is flanked by corresponding GoldenGate sequences to uptake the pMBP1 part in the Golden Gate reaction.(B) Gel electrophoresis indicates successful amplificationof pMBP1. (C) Left: Successful assembled DNA gives rise towhite colonies, while the residual acceptor vector yields red colonies.Right: Negative control, which contained only the acceptor vector in theGolden Gate reaction, yielded only red colonies. (D) Sequencingverification of both assembly junctions shows 100% assembly accuracy.(E) Cost-effectiveness and assembly efficiency comparisonof different reaction volumes for Golden Gate assembly.
Mentions: With Golden Gate assembly, we successfully assembled DNA at a 50 nL reaction volume(typically 15 µL reactions when performed manually), and at the 250 and 500 nLscales the assembly efficiencies are higher than those of the manual control. Thisleads to at least a 30-fold reduction in reagent use when performing Golden Gatereactions using Echo. We did observe vector background in the assembly (redcolonies, as shown in Fig.3C). There are several ways we can overcome this problem.First, instead of using RFP for screening, we can use the toxic ccdB gene, whichcannot give rise to background colonies in a nonpermissive transformation host.Second, we can add a higher concentration of the BsaI enzyme in the Golden Gatemaster mix to further digest the residual acceptor vector. Finally, we may be ableto reduce the background by extending the BsaI digestion step in the incubation.

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.