**6. Conclusions**

genes can be subjected to rounds of *in vitro* evolution, such as error-prone PCR [96] and/or

We demonstrated the first proof-of-concept for designing LOO-GFP biosensors by combin‐ ing computational protein design and *in vitro* evolution. DEEdesign was used to create a set of degenerate oligonucleotide primers for gene assembly. DNA shuffling was performed di‐ rectly on this set of genes to further increase the diversity of the constructed library, since gene assembly mutagenesis does not ensure complete representation of all possible antici‐ pated sequences [94]. DNA shuffling also introduces random mutagenesis beyond the pre‐

Potential candidates for LOO-GFP biosensors were plate-screened in *E. coli* that co-expressed the biosensor gene library and the HA peptide fused to a carrier, intein. Expression of both peptide and biosensor library were induced simultaneously, and the intensity of fluorescence was monitored under excitation of 488 nm wavelength after the induction of 24 hours at room temperature. Two potential LOO-GFP biosensors, DS1 and DS2, that produced elevated fluo‐ rescence intensity in the presence of the HA peptide were found (Figure 12). There were nine and sixteen mutations found in DS1 and DS2 respectively, and seven of those mutations were

**Figure 12.** Potential LOO-GFP biosensors against HA target peptides of influenza virus. (A) Time course study of fluo‐ rescence recovery upon expression of biosensor variants with [+] and without [-] HA peptides. Protein expression was induced with 0.5mM IPTG and under room temperature. Fluorescence was record every hour for 4 hours and after 24 hours. All pictures were taken with the same setting of digital camera. (B) Multiple sequence alignment of LOO7, DS1 and DS2 mutant. Mutations introduced by computational design (green) and *in vitro* evolution (red) in DS1 and DS2

When co-expressed with the HA peptide, the DS1 mutant exhibited target-dependent matu‐ ration of chromophore, while in the absense of the peptide it showed barely detectable fluo‐ rescence even after 24 hours, indicating a specific interaction between DS1 mutant and the HA peptide. DS2 mutant showed faster recovery of fluorescence within four hours in the presence of the HA peptide; however, a higher degree of nonspecific auto-fluorescence was also observed after 24 hours. The DS1 mutant chromophore formation showed a greater de‐ pendency on the left-out peptide (i.e. the HA peptide), implying better folding of designed LOO-GFP molecule, than DS2 mutant *in vivo*, showing DS1 mutant as a better HA-specific

from DEEdesign prediction and the remainder were from *in vitro* evolution.

DNA shuffling [24].

26 State of the Art in Biosensors - General Aspects

mutants are shown.

LOO-GFP biosensor.

dicted mutations on the gene variant.

The unique physical properties of GFP have made it a gold mine for the development of bio‐ sensors and biomarkers. GFP is kinetically super-stable. Its sequence may be readily per‐ muted and mutated. Its engineered variants fluoresce at wavelengths across the visual spectrum, and some pairs of variants can interact via FRET. GFP is quenched by unfolding, by certain ions, and sometimes by light, and variants of GFP are pH sensitive. With many ways of generating a signal, it is no surprise that many types of biosensors have been devel‐ oped that use GFP and its homolog fluorescent proteins.GFP and its variants can be immo‐ bilized and even dried while retaining structure and biosensor function, leading to the promise of future GFP-biosensor microarrays capable of detecting a wide variety of analytes in a single assay. In addition to being broadly useful, such material should be very cheap to produce, and would also be easily stored, used, and read.Arrays of GFP-based biosensors on paper or film may someday become available for household use, so that infections may be rapidly diagnosed without a trip to the hospital, or may become integral parts of devices that continuously monitor the water and air, making the world a healthier and safer place.
