Part:BBa_K2643005

Human EPO gene with artificial intron (615 bp)

BBa_K2643004

Usage and Biology

Usage

The human Erythropoietin (EPO) gene (BBa_K2643004) (Fuertinger, et al., 2012) is used as model for the 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 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 3 and exon 4 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.

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 our EPO with intron 1 biobrick, we used a plasmid harbouring EPO cds (BBa_K2643004) to insert an intron fragment (from kanamycin resistance DNA fragment) and sequence verify.

Strain construction

Aim

Construct a plasmids harbouring EPO with intron 1 (fragment from Kanamycin resistance gene) in pSB1C3 for cloning and iGEM biobrick submission.

Procedure

EPO biobrick (BBa_K2643004) was PCR amplified with primers forward (5’-catcattggcaacgctaccctccatcctcttccaggcat-3’) and reverse (5’-gatatgaataaattgcagtttgtcgggcagcaggccgtaga-3’). Simultaneously, a fragment of 615 bp from the Kanamycin resistance gene was PCR amplified from pSB1K3 with primers forward (5’-atgcctggaagaggatggagggtagcgttgccaatgatg-3’) and reverse (5’-tctacggcctgctgcccgacaaactgcaatttattcatatc-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 (5’-tgccacctgacgtctaagaa-3’) and reverse (5’-attaccgcctttgagtgagc-3’) that target any integration site in pSB1C3. Colony PCR resulted in six possible colonies with correct integration of EPO with intron 1 in pSB1C3 (figure 1, lanes 1, 2, 3, 4, 7, and 8).

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

Transformants 1 and 2 were grown overnight in liquid media and their isolated and purified plasmids were sequence verified. Glycerol stocks of transformant 1 were stored at -80 ºC and its plasmid isolated for further use of the biobrick.

Source

This DNA fragment was synthesised and obtained from EPO biobrick (BBa_K2643004) and the intron sequence was PCR amplified from plasmid backbone pSBiK3.

Safety

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