Part:BBa_K627001

mdnA gene encoding the precursor peptide of the tricyclic microviridin

The BioBrick mdnA is a part of the whole microviridin gene (mdn) cluster, which encodes the protease inhibitor microviridin L. Microviridins are tricyclic depsipeptides, which are ribosomally synthesized by Microcystis aeruginosa (Ziemert et al., 2010). They have a promising potential for therapy as they can block disease-relevant proteases (Ziemert et al., 2008).

Microviridins are synthesized from a ribosomal precursor peptide (MdnA). Additionally, the microviridin L biosynthesis gene cluster consists of genes encoding an ATP-grasp-type ligase (mdnB and mdnC) and genes, which encode an ABC transporter (mdnE) and a N-acetyltransferase of the GNAT family (mdnD) (Ziemert et al., 2008).

The following BioBrick mdnA encodes the ribosomal precursor peptide (MdnA), which is essential for microviridin production (Ziemert et al., 2008).

Because this BioBrick is an RFC10 expression part, the adenin of mdnB gene start codon is part of the XbaI recognition site.

Usage and Biology

Microviridin production in E. coli cells expressing the mdn genes was monitored by reverse phase HPLC. Qualitative analysis involves running a standard that contains the target analytes. The vectors pARW071 and pARW089 served as a control in our experiments because these vectors contain the original mdn-cluster. We could use the retention time as a way to determine the presence of the microviridin production in other samples. HPLC analysis of the purified compound yielded a high peak with a retention time of approximately 5 min. Minor peaks could be detected during the following 7 min (Fig. 3).

Figure 3: HPLC chromatogram of mdnA

All HPLC chromatograms of the isolated mdnA showed reliable peaks with a retention time of 5 min together with a number of following minor peaks.

Fractions of the peaks were sampled and the identity of microviridin and also the presence of cyclization, which is important for the activity, was affirmed with mass spectrometry (Fig. 4). By using a mass spectrometer it is possible to determine both the elemental composition of a fraction and the chemical structure of molecules.

Figure 4: Mass Spectrometry Data

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 150
  
• 1000
  COMPATIBLE WITH RFC[1000]