Part:BBa_K2643009
Human EPO gene with mutations 2 (12 bp)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Usage
The human Erythropoietin (EPO) gene (BBa_K2643004) is used as model for the detection of gene doping by detecting an artificial intronless EPO gene spiked in several samples. The iGEM team from TU Delft (2018) used a fusion protein (BBa_K2643000) to target exon-exon junctions of gene doping EPO gene. One of the sgRNAs used for this fusion protein targeting activity was present at position 148 bp (PAM sequence on reverse strand). To understand the off-target versatility of dxCas9 (part of the fusion protein), a mutant with 12 bp mutations at the end of the guide target was generated.
This sequence can be tested to assess the required specificity between sgRNA and target DNA of Cas9 used to target exon-exon junction of EPO gene at position 148 bp.
Figure 1. Differences between a native and a gene doping gene for EPO. Junctions between exons showed in arrows are the target of detection methods.
Biology
This biobrick can be cloned into Escherichia coli DH5α. After plasmid purification, either PCR amplification of the fragment of interest or its direct use into samples for testing detection methods is recommended.
This biobrick (BBa_K2643009) is only intended for its use to determine specificity parameters of dxCas9.
Characterization
Introduction
In order to characterize our EPO with mutation 2 BBa_K2643009, we used a plasmid harbouring EPO cds (BBa_K2643004) to insert mutations via directed mutagenesis.
Strain construction
Aim
Construct a plasmids harbouring EPO with mutations 2 (12 mutations) in pSB1C3 for cloning and iGEM biobrick submission.
Procedure
EPO biobrick ([https://parts.igem.org/Part:BBa_K2643004 BBa_K2643004) was PCR amplified with forward (5’-ggaggccgagaatatcgtagtaaatcacgctgaacattgcag-3’) and reverse (5’-ctgcaatgttcagcgtgatttactacgatattctcggcctcc-3’) primers containing the desired mutations. The PCR product was digested with DpnI (NEB) prior to direct transformation into chemically competent Escherichia coli BL21(DE3) cells via heat shock (cells containing recombinase system).
Two colonies of transformation were grown overnight in liquid media and plasmid was sequence verified. Glycerol stocks of cells from colony 2 were stored at -80 ºC and plasmid isolated for further use of the biobrick.
Source
This DNA fragment was derived from EPO biobrick (BBa_K2643004).
Safety
We wanted to demonstrate the in vitro functionality of our construct on potential gene doping. As a case study, we focused on the human Erythropoietin gene EPO. The sequence we work with lacks all introns, as this is the scenario for gene doping DNA. The fusion construct is guided to gene doping DNA by gRNAs that target exon-exon junctions which are normally not present in native human DNA. Therefore, the gRNAs we use to target EPO from Homo sapiens would be regarded as safe and harmless. As a confirmation, we received approval from the iGEM Safety and Security Committee to work with these sgRNAs.
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