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All transformants that contained the co-transformed gluc-construct in their genomes were analyzed for functional expression of the luciferase enzyme.
The gluc cDNA fragments were obtained from transformants that contained the chimeric gluc genes following co-transformation with the pPsaD-GLuc, pHRLucP, and pHsp70A-GLuc plasmids.
All transformants that contained both intact aphVIII and g-luc genes were assayed for functional expression of luciferase.
Transformants that contained a gene of the correct size by PCR were then checked using restriction enzyme digestion and DNA sequence analysis.
Transformants that contained a gene of the correct size by PCR were then confirmed as correct by restriction enzyme digestion and by DNA sequence analysis.
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Addition of an enhancer containing three binding sites for the eye enhancer-activator protein Glass (GBS) to pCaSpeR darkens the eye color of transformants that contain an en PRE (Americo et al. 2002).
Forty independent Gonium transformants were propagated in medium that contained the antibiotic paromomycin (1 μg/ml) for two years (or longer), which corresponds to over 1400 colony generations.
Transformants that containing a insert of the correct size were then confirmed as correct by restriction enzyme digestion and by DNA sequence analysis.
The two plasmids were used to co-transform the selection strain US0 by electroporation [31], and co-transformants were selected using medium that contained 3-AT, chloramphenicol (30 µg/mL), kanamycin (25 µg/mL), and tetracycline (20 µg/mL).
Transformants were selected on YPD plates that contained zeocin (300 µg/ml).
Transformants were screened on Murashige and Skoog (MS) medium (M urashige and S koog 1962) that contained 50 μg/ml kanamycin and 100 μg/ml ampicillin.
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