Our team aimed to create three new applications for smURFP. smURFP is a har-infrared fluorescent protein studied by Mr. Qian. small ultra-red FP (smURFP) covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670-nm excitation–emission peaks, a large extinction coefficient (180,000 M-1cm−1) and quantum yield (18%), and photo stability comparable to that of eGFP. smURFP has significantly greater BV incorporation rate and protein stability than the bacteriophytochrome (BPh) FPs. moreover, BV supply is limited by membrane permeability, and smURFPs (but not BPh FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence comparable to that of FPs from jellyfish or coral.
The hypothetical smURFP structure is as follows
With the connection of smURFP and BV, the fluorescence would be detected.
Direction 1: BV Detection
smURFP can connected with biliverdin and fluoresce which has really good transmission. Therefore we can use this protein to detect biliverdin content. Actually it’s really important for birds, reptiles and egg coloration studies. Birds and reptiles have little biliverdin reductase activity and produce very low amounts of bilirubin. Increased concentrations of biliverdin in serum, bile, or excreta can point to anemia, liver problems and biliary obstruction. Biliverdin is also an important pigment of egg shells in several species. Egg coloration studied also require a biliverdin assay to determine biliverdin content in eggshells.
To achieve our aim, we design to set up the relationship between BV, smURFP and fluorescence intensity (RFU). We design our experiments of different concentration of them both and gather their RFU results. Then we draw the data as a series of standard curves. Once we get the relationship, we can use these standard curves to calculate the value of BV concentration that we want when we use proper amount of smURFP to react with the sample. Because of the high cost of biliverdin, we have also tried to produce biliverdin in our lab. To produce biliverdin，the gene of the HO-1 was connected to the expression vector---pET28BS and then transferred to target bacteria E.coli BL-21.The precursor of biliverdin----HEME was transferred to biliverdin with the help of HO-1.After cultured in shaking table, Culture medium turned from yellow to green which proved that biliverdin was produced successfully.
Direction 2: Intestinal Bacteira Tracking
To reach our goal, we select two methods, co-expression and surface display. To construct the co-expression system, the gene of fluorescent protein---smURFP and the gene of the precursor of biliverdin---HO-1 should be connected to the same expression vector and then transferred to our target bacteria. The precursor of biliverdin will be transferred to biliverdin through a series of conversion, and then fluorescent protein will combine with biliverdin directly in our target bacteria and glow in the bacteria. Because there’s no oxygen in anaerobic bacteria, so the co- expression system won’t work in the anaerobic bacteria.
To construct the surface presentation 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.
The strains and plasmids we choose could be noticed at the table.
|Bifidobacterium longum||pGH||Anaerobic bacteria|
|Citrobacter rodentium||pACYC184||facultative anaerobes|
|enterohemorrhagic E.coli||pACYC184||facultative anaerobes|
The module construction of Co-expression system:
The module construction of surface presentation system:
Direction 3: Protein Strucute Measuring
To make the protein better, there are two choices. One is random mutation, which was done by Mr. Tsien, who studied it for 10 years and finally got the target protein. The other is accurate redesign, and precise mutation for active site will greatly shorten the period. It is obvious to us that the second methods is better, which means it require the structure of the protein. Following this idea, we have designed the process of protein purification. Three steps are there for purification: nickel column, ion exchange column and molecular sieve column with AKTA system.
 Rodriguez EA, Tran GN, Gross LA, Crisp JL, Shu X, Lin JY, Tsien RY. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein. Nat Methods. 2016 Sep;13(9):763-9.