Encoding and Expression of smFPs

DNA encoding smFPs were ordered from DNA2.0. Genes encoding smFPs were sub-cloned into pRSETa (Life Technologies) for protein expression and purification in Escherichia coli BL21 (this adds an N-terminal His tag for purification, and increases the MW by 4 kDa). Genes encoding smFP variants were sub-cloned into the pCAGGS vector with a CAG promoter (CMV enhancer, β-actin promoter) and regulatory element from the woodchuck hepatitis virus (WPRE)^{50 (link)} for expression in HeLa cells and in utero electroporation^{51 (link), 52 (link)}. For expression in flies, R59A05-GAL4^{49 (link)} was used to drive expression of UAS-smFP reporter constructs. For expression in mice, GFP and smFP variants were expressed using an adeno-associated virus serotype 2/1 (AAV2/1) driving the probe under control of the human synapsin-1 promoter or a Cre-dependent (FLEX) version of the CAG promoter; live virus was produced (Janelia Farm Viral Vector Core). All constructs were verified by sequencing.

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Viswanathan S., Williams M.E., Bloss E.B., Stasevich T.J., Speer C.M., Nern A., Pfeiffer B.D., Hooks B.M., Li W.P., English B.P., Tian T., Henry G.L., Macklin J.J., Patel R., Gerfen C.R., Zhuang X., Wang Y., Rubin G.M, & Looger L.L. (2015). High-performance probes for light and electron microscopy. Nature methods, 12(6), 568-576.

Publication 2015

pRSETa

Actin Adeno associated virus 2 Dna2 Escherichia coli Flies Genes Hela cells Human Mice Protein Regulatory element Synapsin 1 Utero Vector Virus Woodchuck hepatitis virus

Corresponding Organization :

Other organizations : Janelia Research Campus, Howard Hughes Medical Institute, University of Utah, Harvard University, National Institute of Mental Health

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Protocol cited in 14 other protocols

Variable analysis

independent variables

Different smFP variants expressed in cells/organisms

dependent variables

Expression of smFP variants in E. coli, HeLa cells, flies, and mice
Protein purification of smFP variants from E. coli

control variables

N-terminal His tag added to smFPs expressed in E. coli
PRSETa vector used for smFP expression in E. coli
PCAGGS vector with CAG promoter used for smFP expression in HeLa cells and in utero electroporation
R59A05-GAL4 driver used for smFP expression in flies
AAV2/1 virus with human synapsin-1 promoter or Cre-dependent CAG promoter used for smFP expression in mice

controls

Positive control: GFP expressed in mice using AAV2/1 virus
Negative control: Not explicitly mentioned

Annotations

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