Part:BBa_K2643006

Human EPO gene with artificial intron (135 bp)

Usage and Biology

Usage

The human Erythropoietin (EPO) gene (BBa_K2643004) is used as model for detection of gene doping in samples spiked with an artificial intronless EPO gene. This biobrick contains an insertion of 135 bp at position 426 bp of EPO cds to mimic the presence of a large intron at this position.

This sequence can be tested to asses and replicate the results on targeted sequencing with the fusion protein dxCas9-Tn5 (BBa_K2643000) generated by iGEM TU Delft 2018. This biobrick can be used to prove the specificity of a RNA guided Cas9 protein towards the junction between exon 4 and exon 5 of EPO cds. This biobrick can further be used for the development of new detection methods of gene doping, together with EPO cds as gene doping model.

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 is only intended for its use as contaminant transgene in DNA samples to test for gene doping.

Characterization

Introduction

In order to characterize our EPO with intron 2 biobrick, we used a plasmid harboring EPO cds (BBa_K2643004) to insert an intron fragment (from ampicillin resistance DNA fragment) and sequence verify.

Strain construction

Aim

Construct a plasmids harbouring EPO with intron 2 (fragment from Ampicillin resistance gene) in pSB1C3 for cloning and iGEM biobrick submission.

Procedure

The EPO biobrick (BBa_K2643004) was PCR amplified with primers forward (5’-aatagactggatggaggcggctgggctcccagagcccgaa-3’) and reverse (5’-cgtttggtatggcttcattcaaggaagccatctcccctcc-3’). Simultaneously, a fragment of 135 bp from Ampicillin resistance gene was PCR amplified from pSB1A3 with primers forward (5’-ttcgggctctgggagcccagccgcctccatccagtctatt-3’) and reverse (5’-ggaggggagatggcttccttgaatgaagccataccaaacg-3’). Both products were assembled via Gibson Assembly (Gibson Assembly Master Mix NEB), and transformed into chemically competent E. coli DH5α cells via heat shock.

Transformed cells were screened via colony-PCR using primers forward VF2 (5’-tgccacctgacgtctaagaa-3’) and reverse VR (5’-attaccgcctttgagtgagc-3’) that target any integration site in pSB1C3. Colony PCR resulted in three possible colonies with the correct integration of EPO with intron 2 in pSB1C3 (figure 1, lane 1, 3 and 4).

Figure 1. Colony PCR of EPO with intron 2. The ladder represents the size of DNA in bps.

Transformant 1 was grown overnight in liquid media and its plasmid was subsequently isolated, purified and sequence verified. Glycerol stocks of cells from transformant 1 were stored at -80 ºC and plasmid isolated for further use of the biobrick.

Source

This DNA fragment was synthesized was obtained from EPO biobrick (BBa_K2643004) and the intron sequence was PCR amplified from plasmid backbone pSBiA3.

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 gRNAs.