Team:TJU China/Demonstrate
Demonstration
This year we have used a novel fluorescent protein, smURFP, for three kinds of different usage. The first is BV detection. We have construct a series of standard curves among concentration of BV and smURFP and its relative fluorescent units (RFU). Then we can use different amount of smURFP to test the sample, and get the final concentration of BV through the curves. The second is intestinal bacteria tracking. We have constructed series of corresponding plasmids of co-expression system and surface display system for different intestinal bacteria. And now we can be sure that our construction can work in vivo with very competible persistence and penetrability. The third is structure analysis of smURFP. We successfully gain the 3D real structure of the protein. Then anyone can use this structure information for further study and mutation.
1. BV production
Our aim is to test the accurate amount of BV using smURFP we produced and purified. Then we get a series of standard curves of different concentration of BV and smURFP, as shown in the text-center below. Then we can use these curves to calculate how much BV is there in birds’ blood, or in other solutions.
Form the results, we found that we can get good standard curves if we use more than 15μM (15μM included) protein for detection. And from these four curves, we got a final standard curve, as shown in the figure below.
Figure. Standard curves between concentration of BV and RFU. (concentration of smURFP should be more than 15μM)
2. Intestinal bacteria tracking
2.1. Bacteria tracking in vitro
Escherichia coli BL21 - Co-expression
Figure 2. Laser confocal microscopy was use to observe these bacteria directly. Excitation wavelength is 640nm.
2.2. Bacteria tracking in vivo
Escherichia coli BL21 - Co-expression
Figure 3. The picture shows relative fluorescent intensity after doing intragastric administration (108 and 1011 CFU) for 5.5h. The left is the control one (with E.coli without plasmid), the right is the experimental one (with E.coli with plasmid pET28b, 1011 CFU). The result successfully showed that our system was executable and excellent. And smURFP has very compatible persistence and penetrability.
Figure 4. Trend of RFU in vivo. M2 is the mice with 108 CFU bacteria, and M3 is the mice with 1011 CFU. We can see the fluorescence is relatively high, and the RFU is still over 3000 after 300min when the mice with 1011 CFU bacteria.
