Part:BBa_K2643007

Human EPO gene with 2 artificial introns (615 + 135 bp)

BBa_K2643007

Usage and Biology

Usage

The gene (BBa_K2643004) encoding for the human erythropoietin (EPO) (Fuertinger, et al., 2012) . is used as model for detection of gene doping in samples spiked with an artificial intronless EPO gene. This biobrick contains an insertion of 615 bp at position 246 bp of EPO and another insertion of 135 bp at position 426 bp of EPO cds to mimic the presence of two different introns in EPO.

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 junctions between exons 3, 4 and 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. Detection methods for gene doping target the junction between exons of the DNA sequence used for gene transfection (Figure 1):

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 DH5 α. 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Introduction

In order to characterize the biobrick EPO with introns 1 and 2, we used a plasmid harbouring EPO with intron 1 cds (BBa_K2643005) to insert another intron fragment (from ampicillin resistance DNA fragment) and sequence verify.

Strain construction

Aim

Construct a plasmids harbouring EPO with introns 1 and 2 (fragments from Ampicillin and Kanamycin resistance genes) in pSB1C3 for cloning and iGEM biobrick submission.

Procedure

The EPO with intron 1 biobrick (BBa_K2643005) was PCR amplified with primers forward (5’-cgtttggtatggcttcattcaaggaagccatctcccctcc-3’) and reverse (5’-aatagactggatggaggcggctgggctcccagagcccgaa-3’). Simultaneously, a fragment of 135 bp from the Ampicillin resistance gene was PCR amplified from pSB1A3 with primers forward (5’-ttcgggctctgggagcccagccgcctccatccagtctatt-3’) and reverse (5’-ggaggggagatggcttccttgaatgaagccataccaaacg-3’). Both products were ligated via Gibson Assembly (Gibson Assembly Master Mix NEB), and transformed into chemically competent Escherichia 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 correct integration of intron 1 and intron 2 in EPO (EPO with both introns) in pSB1C3 (figure 2, lanes 2, 3 and 7).

Figure 2. Colony PCR of EPO with intron 1 and intron 2. The ladder represents the size of DNA in bps. Lanes 2, 3 and 7 show the apparent correct integration of intron 2 into EPO with intron 1, with an expected size of 1100 bp.

Transformant 2 was grown overnight in liquid media. Subsequently, its plasmid was isolated, purified and sequence verified with primers VF2 and VR. Glycerol stocks of cells from transformant 2 were stored at -80 ºC and more plasmid was isolated for further use of the biobrick.

Source

This DNA fragment was derived from EPO with intron 1 biobrick (BBa_K2643004) and the intron sequence was PCR amplified from plasmid backbone pSBiA3.

Safety

This part can be used in BSL-1 biosafety level laboratory.