Team:DTU-Denmark/Description
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Improvement and characterization of two existing parts
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Improvement and characterization of two existing parts We have improved two existing BioBricks by successful removal of two illegal restriction sites and further improved the characterization.
In our laboratory work, we wanted to work with the two BioBricks; BBa_K530001 (CrtE gene) and BBa_K530002 (CrtI gene) created by the John Hopkins iGEM team 2011. The genes encode the two enzymes Geranylgeranyl Diphosphate Synthase and Phytoene Desaturase, respectively, both from the wildtype strain of xanthophyllomyces dendrorhous used in the biosynthesis of B-Carotene.
When looking into the specifications of these BioBricks, we realized that both genes contained illegal restriction sites. BBa_K530001 contained an AgeI restriction site making the part incompatible with the RFC25 Freiburg Standard. The RFC25 standard allows for in-frame assembly of protein domains. BBa_K530002 contained the illegal restriction site BglII making the part incompatible with the RFC21 Berkely Standard, which enables in-frame assembly of proteins.
We wanted to overcome these obstacle by removing the illegal restriction sites from the above described Biobricks.
How did we do it?
The illegal restriction sites were removed using site directed mutagenesis with primers containing nucleotide substitutions in the two restriction sites (see figure. Protocol https://static.igem.org/mediawiki/2014/b/b5/Wageningen_UR_protocols_Site_Directed_Mutagenesis.pdf).
Text for figure:Primers overlapping the restriction sites are designed with a single nucleotide change to disrupt the restriction site. The primers anneal to the template plasmid and replicate while introducing the point mutation. The elongated plasmids are digested with the enzyme DpnI, which cleaves at the methylated sites breaking down the circular template, resulting in a higher transformation efficiency of the linear PCR product.
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Description of biobrick
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Description of biobrick
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Section 5
Has ut facer debitis, quo eu agam purto. In eum justo aeterno. Sea ut atqui efficiantur, mandamus deseruisse at est, erat natum cum eu. Quot numquam in vel. Salutatus euripidis moderatius qui ex, eu tempor volumus vituperatoribus has, ius ea ullum facer corrumpit.
