Part:BBa_K2643008

Human EPO gene with mutation 1 (5 bp)

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]

Usage and Biology

Usage

The Human Erythropoietin (EPO) gene (BBa_K2643004) (Fuertinger, et al., 2012). was used by iGEM TU Delft team 2018 as a model for the detection of gene doping by detecting an artificial intronless EPO gene spiked in serum samples. The mentioned team 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 of the EPO gene (PAM sequence on reverse strand). To understand the off-target versatility of dxCas9 (part of the fusion protein), a mutant EPO gene with 5 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 the exon-exon junction at position 148 bp of the EPO gene. 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 transformed 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 to determine specificity parameters of dxCas9.

Characterization

Introduction

In order to generate the BBa_K2643008 biobrick, we used a plasmid harbouring EPO cds (BBa_K2643004) to insert mutations via directed mutagenesis.

Strain construction

Aim

Construct a plasmid harbouring EPO with mutations 1 (5 mutations) in pSB1C3 for cloning and iGEM biobrick submission.

Procedure

The EPO biobrick (BBa_K2643004) was PCR amplified with forward (5’-ggaggccgagaatatcacgacggatcacgctgaacattgcag-3’) and reverse (5’-ctgcaatgttcagcgtgatccgtcgtgatattctcggcctcc-3’) primers containing the desired mutations. The PCR product was digested with DpnI (NEB) prior direct transformation into chemically competent E. coli BL21 DE3 cells via heat shock (cells containing recombinase system). This is necessary so the cells can do homologous recombination of the complementary flanks of transformed DNA.

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

Source

This DNA fragment was derived from the EPO biobrick (BB BBa_K2643004).

Safety

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