The rate of activation, demonstrated by the time constant of activation (τact) was accelerated to the same extent by the two RFP-Sx1A proteins (Fig. 2A,B lower) consistent with the untagged Sx1A [23], [23].
DOI: http://dx.doi.org/10.7554/eLife.05007.022 10.7554/eLiFigure07.023 Figure 8 figure supplement 1. Cartoons of lineages that showed two RFP-to-GFP switches in directly related cells.
In the present paper, we describe the combined use of rational design and directed evolution for the engineering of two RFP-based Ca2+ indicators for imaging of Ca2+ dynamics within mitochondria and ER.
In contrast, 8 of the 12 cells that expressed detectable GFP as large-budded cells gave rise to mother-daughter pairs in which only the mother or daughter remained green (patterns 7 and 8), indicating that Cre catalyzed recombination after S phase and acted on only one of the two RFP-GFP cassettes.
In a second round of pathogenicity assays we compared IPO323 against the four RFP expressing strains at 16 dpi.
To this end, three RFP- red fluoRFP- redprotein)-marked insertions at positions 62B, 85E and 92A were used as genetic markers (one ofluorescentted close to Df(3R)Exel6149 at protein -markedhich was isolated as a dominant suprotein -markedhe definsertionsreen).
(A ) Representative florescent images showing that elo-5 animals with each of four RFP-marked transgenes, which constitutively activated TORC1, bypass L1 arrest to reach beyond L3 stage (statistical data are described in the text and Figure 4B ).
Among five RFP-transgenic positive individuals, there were one to four genomic integration sites in the genome of the blunt snout bream, including the specific end sequences of Thm1 DNA (Table 3; Figure S1), whereas no PCR products during splinkerette PCR were amplified in the genome of the negative control (data not shown).
Eight sites (51MZQ, 51WCW, 51SFB, 51JYD, 51JYC, 51JYH, 51GYZ, and 51GYS) could be identified using any of the four parameters, RFp, DNL, ADNL, and PNL, whereas the other eight sites (51AXT, 51JZW, 51XJD, 51LXT, 51MXN, 51TQL, 51JZB, and 62WUD) could be identified using individual parameters.
To engineer new Ca2+ sensors with decreased affinity, we initially considered two single RFP-based Ca2+ indicators, R-GECO1 (Kd=0.48 μM) [ 5] and R-GECO1.2 (Kd=1.2 μM) [ 6], as potential templates.
(B ) Four representative RFP-expressing granule cells 34, 47, and 63 days after CAG-RFP virus labeling.
