Part:BBa_K2643004

Coding region (CDS) of human erythropoietin (EPO) hormone BBa_K2643004 Coding sequence of human Erythropoietin hormone cds.

Usage and Biology

Usage

The human Erythropoietin (EPO) hormone is a precursor of red blood cells. In mammalians EPO is generated in kidneys and travels through the bloodstream to the bone marrow to stimulate red blood cell formation (Fuertinger, et al., 2012). The EPO DNA sequence is used as model for gene doping detection. With EPO of the usage of gene doping with EPO transgenic gene by athletes. It was used to assess and validate the results on targeted sequencing with the fusion protein dxCas9-Lin-Tn5 (BBa_K2643000) created by iGEM TU Delft 2018. This biobrick can further be used to develop new detection methods of gene doping, using EPO gene as a reference and case study gene.

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 as contaminant transgene in DNA samples to model gene doping detection. This part is not intended for the expression of EPO hormone (Fuertinger, et al., 2012).

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 biobrick, we sequence inserted in a plasmid (XXX), sequence verified and analysed its presence in serum samples.

Strain construction

Aim

Construct a plasmids harbouring EPO coding sequence in pSB1C3 for cloning and iGEM biobrick submission.

Procedure

EPO cds was ordered as a G-Block from IDT with extremes for restriction ligation into pSB1C3 backbone from the iGEM competition kit. Upon arrival, primers forward (5’-GATCGAATTCGCGGCCGCTTC-3’) and reverse (5’-CGATTCTGCAGCGGCCGCTAC-3’) were used for PCR amplification of G-block, followed by restriction with enzymes EcoRI HF and PstI HF and ligation with T4 ligase (all reagents from New England Biolabs) and pSB1C3 restricted under same conditions. The ligate product was transformed into chemically competent E. coli DH5α cells and transformed 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 three possible colonies with correct integration of EPO in pSB1C3 (figure 1, lane 1, 2 and 3) with an expected size of 824 bp.

Figure 1. Colony PCR of EPO. The ladder represents the size of DNA in bp.

Transformants 1 and 2 were grown overnight in liquid media (LB) and plasmids were isolated, purified and sequence verified with the use of primer forward (5’-tgccacctgacgtctaagaa-3’) (Ez-seq). Glycerol stocks of transformant 2 were stored at -80 ºC and its plasmid isolated for further characterization of the biobrick.

Source

This DNA fragment was synthesised with Integrated DNA Technologies (IDT). The sequence was retrieved from NCBI (GenBank: BC143225.1).

Safety

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