Part:BBa_K2328000
smURFP (I, codon-optimized for Escherichia coli) (without terminator codon TAA)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage
smURFP (small ultra-red FP) is an important part in our group. It is desirable for our BV detection and in-vivo imaging because with it molecule less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow and orange FPs. smURFP can covalently attaches a biliverdin(BV) chromophore without a lyase, and has 642/670 nm excitation - emission peaks, a large extinction coefficient and quantum yield, and photostability comparable to that of eGFP.
Biology
In order to fluorescence, smURFP must be combined with biliverdin (BV) .So we construct the surface display system to make in-vivo imaging come true. To construct the surface display system, the gene of fluorescent protein---smURFP and the gene of the anchoring protein should be connected to the same expression vector. After the recombinant plasmid is transferred to the target bacteria, the fluorescent protein and anchoring protein will express at the same time and become fusion protein, and then the fluorescent protein will be carried to the cell surface by anchoring protein. With the added biliverdin, fluorescent protein will combine with biliverdin and glow on the cell surface.
Reference
[1] Rodriguez EA,Tran GN , Gross LA, et al. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein .[J].NATURE METHODS,2016:763-769.
Results
We did some experiments on smURFP and BV.
Protein and BV
We test the fluorescence changes of different concentration of smURFP and BV and the fluorescence.
Figure 1. A standard curve of fluorescence intensity changes of different concentration of smURFP and BV.
Surface display system in E.coli BL21 (in vitro)
The laser confocal microscopy was use to observe these bacteria, activate light of 640nm was used, as shown in Figure 2.
Figure 2. The result after induction, as we can see, fluorescent protein combine with biliverdin and glow on the cell surface.
Surface display system in E.coli BL21 (in vivo)
After tests in vitro, we used this engineered bacteria for experiments in vivo.. Utilizing Animal imaging system, we consistently observed the fluorescence emitted from the bacteria in mices' gut. The result successfully showed that our system was executable and excellent. And smURFP has very competible persistence and penetrability.
<p style="text-align: center;">
Figure 3. The fluorescent inensity after doing intragastric administration for 5.5h.The left is the control one, the right is the experimental one.
After the experiment, we gathered the data of RFU and drawed a gragh about its trend.
Figure 4. Trend of RFU in vivo. M2 is the mice with 10^8 CFU bacteria, and M3 is the mice with 10^11 CFU. We can see the fluorescence is relatively high, and the RFU is still over 3000 after 300min when the mice with 10^11 CFU bacteria.
| None |