Part:BBa_K2117012
CrtE improved
Improved version of biobrick encoding the CrtE enzymes, which catalyses a step in the beta-carotene pathway. Removing an illegal restriction site making the Biobrick compatible with the RFC25 standard for in-frame protein fusion.
The JHU 2011 iGEM team produced beta-carotene in Saccharomyces cerevisiae by constructing three biobricks with the three individual genes encoding the enzymes from the pathway from the fungi Xanthophyllomyces dendrorhous.
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 Beta-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. 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).
Figure 1: 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.
Table 1: Primers designed for removal of illegal restriction sites. Bold marks the nucleotide substitution.
The new plasmid, BBa_K2117012, with the removed AgeI restriction site was double digested with the enzyme and SpeI to test if we had successfully removed the AgeI restriction site.
Figure 2: AgeI + SpeI digestion. Electrophoresis on a 1% agarose gel showing the digestion of BBa_K2117012 (lane 1-3). BBa_530001 digested with AgeI + SpeI and undigested BBa_530001 were used as controls (lane 4+5).
The digestion showed the removal of the AgeI restriction site, shown on the gel picture by only one band on the BBa_K2117012 compared to two bands on the control BBa_K530001.
BBa_K2117012 and BBa_K2117013 were sent for sequencing with the verification primers VR and VF2 primers to further verify the removal of the restriction sites.
Figure 3: Sequencing alignment of BBa_530001 (top sequence) and BBa_K2117012 (bottom sequence). The alignment shows a nucleotide substitution in the AgeI restriction site.
Figure 4: Sequencing alignment of BBa_530002 (top sequence) and BBa_K2117013 (bottom sequence). The alignment shows a nucleotide substitution in the BglII restriction site.
The sequencing results show a substitution in the restriction sites corresponding to the nucleotide substitution designed in the primers (table ??). By deleting the two restriction sites in the BBa_K2117012 and BBa_K2117013 we have made the two BioBricks compatible with RFC25 and RFC21 standards, respectively. Sequencing results of BBa_K530002 showed nucleotide substitutions in the prefix and a large deletion in the suffix.
Figure 5: Alignment of BBa_K530002 sequence received from the parts page (top) and sequencing results of BBa_K530002 from the distribution kit. Alignment shows many nucleotide substitutions in the prefix seuqence.
Figure 6: Alignment of BBa_K530002 sequence received from the parts page (top) and sequencing results of BBa_K530002 from the distribution kit. Alignment shows a deletion in the suffix sequence.
The alterations in prefix and suffix make the BBa_K530002 incompatible with the BioBrick standard
Usage and Biology
Beta-carotene is naturally produced by a range of organisms such as plants and fungi, but neither conventional yeast nor Y. lipolytica has the pathway for biosynthesis. Beta-carotene is produced by four enzymatic steps from farnesyl diphosphate (F-PP), which is naturally produced in Y. lipolytica. In the next step, farnesyl diphosphate is converted to geranylgeranyl diphosphate (GG-PP) in a reaction catalyzed by geranylgeranyl diphosphate synthase (CrtE). GG-PP is transformed to phytoene by CrtYB, which is an enzyme with two domains, one functioning as phytoene synthase and another as lycopene cyclase, in this reaction the first domain plays a crucial role. The next step results in production of lycopene and is catalyzed by carotene desaturase (CrtI). Finally, lycopene is converted by CrtYB with the lycopene cyclase domain into beta-carotene6.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 335
Illegal XhoI site found at 34
Illegal XhoI site found at 166
Illegal XhoI site found at 985 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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