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Yeast one-hybrid assay using the pLacZi2u/pB42AD system

HL Huang Liyu

Last updated date: Apr 8, 2026 Views: 21 Forks: 0

original An abbreviated version of this protocol was published in Nature Communications
Neo-functionalization of a Teosinte branched 1 homologue mediates adaptations of upland rice
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Protocol of yeast one hybrid assay using pLacZi2u/pJG4-5 system

  1. Vectors

pLacZi2μ (AmpR in E.coli; Ura- in yeast) and pJG4-5 (or named pB42AD; AmpR in E.coli; Trp- in yeast) plasmids. 2μ origin of replication was PCR amplified from pEG202 and inserted into the HindIII and EcoRI sites of the reporter plasmid pLacZi (Clontech), resulting in pLacZi2μ vector.

The coding sequence of DNA binding protein should be inserted into the MCS of pJG4-5 to generate the AD-fusion construct and the cis-element to be tested needs to be inserted into the MCS of the pLacZi2μ reporter plasmids.

  1. Yeast strain

Yeast EGY48 strain is used in this system, other strains may also work but need to be tested.

  1. Small scale yeast transformation

Plasmids for GAD fusions were co-transformed with LacZ reporter genes driven by promoter fragments for testing into EGY48. Transformants were grown on SD (galactose+raffinose)/-Ura/-Trp/ X-gal plates. 

 

References:

Science 318, 1302 (2007); Nature cell biology. Online 17 April 2011; DOI: 10.1038/ncb2219

 

Copyright: © 2026 The Author(s); This is an open-access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Liyu, H(2026).

    Yeast one-hybrid assay using the pLacZi2u/pB42AD system

    . Bio-protocol Preprint. bio-protocol.org/prep2923.
  2. Lyu, J., Huang, L., Zhang, S., Zhang, Y., He, W., Zeng, P., Zeng, Y., Huang, G., Zhang, J., Ning, M., Bao, Y., Zhao, S., Fu, Q., Wade, L. J., Chen, H., Wang, W. and Hu, F.(2020). Neo-functionalization of a Teosinte branched 1 homologue mediates adaptations of upland rice. Nature Communications 11. DOI: 10.1038/s41467-019-14264-1

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