Team:DTU-Denmark/Description

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"A great building will never stand if you neglect the small bricks"
Ifeanyi Enoch Onuoha

BBa_K2117000

Constitutive TEF1 promoter

BBa_K2117001

hrGFP codon-optimized for Y. lipolytica

BBa_K2117002

Composite part consisting of the TEF1 promoter and the human proinsulin gene codon-optimized for Y. lipolytica

BBa_K2117003

Human proinsulin codon-optimized for Y. lipolytica

BBa_K2117004

SCR1'-tRNA promoter expressing sgRNA

BBa_K2117005

hrGFP expressed by the constitutive TEF1 promoter

BBa_K2117009

Semi-synthetic shuttle vector pSB1A8YL

BBa_K2117010

Composite part of TEF1 and enhanced YFP

BBa_K2117011

Composite part of TEF1 and enhanced CFP

BBa_K2117012

Improved BBa_K530001(crtE)

BBa_K2117013

Improved BBa_K530002(crtI)

Improvement and Characterization of Two Existing Parts

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.

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 1).

DESCRIPTION — **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.

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.

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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.

The sequencing results show a substitution in the restriction sites corresponding to the nucleotide substitution designed in the primers (table 1).

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.

The alterations in prefix and suffix make the BBa_K530002 incompatible with the BioBrick standard