Part:BBa_K4030000

OmpA

Usage and Biology

Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane.

Experimental approach

OmpA was linked with ClyR. Then, the sequence was inserted into plasmid and we can get recombinant plasmid. Plasmid puc57-kan-mini-J23101-OmpA-araB-TT and plasmid pBAD-Myc-HisA-OmpA-ClyR-6His-TT were co-transformed to E. coli Nissle 1917 by electroporation.

The araboxylan with final concentration 0, 0.3%, 0.6%, 1.0%, 1.5% and 2.0% (w/v) was added into the culture to induce ClyR expression.

For the comparison, E. coli Nissle 1917 with plasmid B was cultured by the same way and the expression of ClyR was induced by the addition of arabinose with final concentration of 0 μM, 10 μM,30 μM,0.1 mM, 0.2 mM,0.5 mM and 2 mM. The protein concentration was monitored at 595 nm using Multiscan Spectrum (BioTek). Read the data for three times, recorded the average of the A595 data.

Gels were scanned with the ImageQuant™ LAS 4000 mini (GE Healthcare).

Proof of function

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Figure 1 The SDS-PAGE of supernatant..

The A595 of the culture supernatant of E. coli with plasmid A and B suggested the possible expression of ClyR (Table 2). The A595 data at “0 %” was referred as blank, and the protein concentration could be calculated referred to the standard formula. As can be seen from figure 1, in the transformed bacteria with plasmid A and B, the concentration of the reducing sugar increased with the elongation of the incubation time.

During the expression process, bacteria lysis occurred with the elongation of the incubation time. But the ClyR band could obviously be obtained as is shown in figure 1.

In the engineering cells containing plasmid A and plasmid B, the expression of ClyR was successful.

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]