Part:BBa_K3332012

INPNC-GRHPR

We anchor GRHPR protein onto membranes through INPNC to catalyze the reaction of reducing glyoxalic acid and consuming NADPH. We use BBa_K880005 to construct the expression system and anchor GRHPR on the surface of E.coli.

Biology

Ice nucleoprotein is an anchor protein from Pseudomonas syringae. It can anchor its passenger protein to the cell membrane. N and C terminal of Ice nucleoprotein, which is named after INPNC, can also anchor passenger protein fused with it to the cell membrane. GRHPR, a glyoxylate reductase from human liver, can reduce glyoxylic acid when NADPH is used as cofactor. GRHPR is fused at N terminal with INPNC so that GRHPR can be displayed on the surface of E. coli.

Fig 1. Mechanism of GRHPR on the surface of E. coli.

Usage

Here, we used BBa_K880005 to construct the expression system and demonstrated the effect of INPNC-GRHPR on the surface of E. coli. We obtained the composite part BBa_K3332057 and transformed the constructed plasmid into E. coli BL21 (DE3) to verify its expression. The positive clones were cultivated.

Fig 2. Gene circuit of INPNC-GRHPR.

Characterization

1. Identification

After receiving the synthesized DNA, PCR was done to certify that the plasmid was correct, and the experimental results were shown in figure 3.

Fig 3. DNA gel electrophoresis of PCR products of INPNC-GRHPR-pSB1C3 (Xbal I & Pst I sites)

2. Ability of consuming NADPH

We mixed glyoxylic acid solution, NADPH solution and bacteria solution carrying INPNC-GRHPR. Then, we immediately measured OD₃₄₀ changes. TECAN^® Infinite M200 Pro was used to detect OD₃₄₀. And when NADPH is consumed, OD₃₄₀ declines.

We successfully got OD₃₄₀-Time curves of GRHPR fused with 4 types of anchor protein. When using INPNC-GRHPR, we could see OD₃₄₀ decreased as the reaction went on. And by using J23100-RBS (BBa_K880005) and GRHPR-Histag as control, we could find that the slopes of experimental and control curves have a significant difference, which means that our fusion protein INPNC-GRHPR is displayed on the surface and works very well.

The result is shown in figure 4.

Fig 4. OD₃₄₀-Time curves of GRHPR fused with 4 types of anchor protein.

References

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 NgoMIV site found at 405
  Illegal AgeI site found at 1522
  
• 1000
  COMPATIBLE WITH RFC[1000]