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.

Insert table

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 text: 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.

insert plasmids

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.

Improvement of Characterization

While working with BBa_K530001 and BBa_K530002 we were struggling with assembly of the genes. Therefore, we sequenced the plasmids received in the distribution kit.

Sequencing results of BBa_K530002 showed nucleotide substitutions in the prefix and a large deletion in the suffix.

figure text 1: Figure text: 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 text 2: Figure text: 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

Parts list

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Section 4

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Section 5

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Section 6

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Section 7

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