Part:BBa_K1378007

INPNC-MVN & GFP (the Binding Plasmid)

Introduction

This plasmid is the final testing plasmid for our E. coli- M. aeruginosa binding assay. We inserted two transcription units into one plasmid and they are expressed under the promoters J23105 and J23117 respectively. After this plasmid is transformed into E. coli, the mannan binding protein MVN (microvirin) will be displayed on the cell surface by INPNC (Ice Nucleation Protein N and C terminal) and the E. coli cell will express GFP as well. In this way, the E. coli cells will be able to bind to M. aeruginosa cells in a "visible" way.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 1495
  Illegal NheI site found at 1518
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 466
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2196
  Illegal SapI site found at 1038
  

Characterization

This binding assay is the final test to identify the binding effect of our genetically modified E. coli. First, we designed and constructed the plasmid for the experiment group (this part) . We also constructed one plasmid for the control group that only had the second transcription unit encoding GFP. After construction, the plasmids were transformed into E. coli BL21(DE3) cells.

We carried out this binding test with M. aeruginosa FACHB-1343. The cells of the experiment group and control group were both incubated till their OD₆₀₀ reached 0.6. Then both E. coli and M. aeruginosa were applied to short period of mild sonication to break up the colony formed and harvested by centrifugation and PBS washing. E. coli and M. aeruginosa were co-incubated and observed under fluorescence microscope afterwards. We used microscopic counting to quantify the binding effect.

The binding assay was carried out with both the experimental group and the control group. Each test was done for several times to make the results persuasive. We randomly chose 18 views for both the experimental group and the control goup. Every view was observed in the blue and green channel respectively and later merged together to see the binding effect. The binding effect of the experimental group is shown below in Fig. 1.

Figure 1.Binding of E. coli to M. aeruginosa observed under the fluorescence microscope. This mosaic is made up of several sectional views taken by the fluorescence microscope. The red dot represents the M. aeruginosa cell and the green rod represents the E. coli cell.

As can be seen from the figure, the binding effect can be observed qualitatively. Later, we used microscopic counting to determine the exact binding ratio. The maximum number of E. coli cells bound to one M. aeruginosa is 4, so we counted the amount of M. aeruginosa cells with 0, 1, 2, 3 and 4 E. coli cells bound. All of the cyanobacteria included in the 18 views were counted, and the result is shown in Fig. 2.

The contrast of binding proportion between the experiment group and control group is notably different. In addition, the difference is even greater when the binding amount reaches 3 or 4, when the random binding is less possible. This result does prove that our design really works and that we can successfully bind our E. coli to M. aeruginosa.

Figure 2. Quantitative binding effect of E. coli to M. aeruginosa.