The entire protein is folded natively, it does show that critical tertiary structure is achieved since the binding of tachykinins has been shown to involve liganding from residues on a least 3 different transmembrane domains. GPCRs involve a greater level of complexity. This includes G-protein activation and receptor internalization, which are more complete Doxorubicin measurements of GPCR function. Because our system also lacks post-translational modifications, cell membrane lipid components, and the heterotrimeric G-protein, we do not expect our assays to fully mirror the protein in a cell membrane. In the future, such studies could be possible using techniques such as FCS within cells. FCS is highly amenable to measurements in solution utilizing cross-correlating measurements, which would potentially allow measurements in heterogeneous environments such as cell membranes and cell fractions. In the future such experiments could be designed to better access both the in vitro and in vivo biology of GPCRs complexed with NLPs. In summary, we applied a de novo synthesis, cell-free coexpression, and in-situ analysis method to produce nanolipoprotein particles capable of solubilizing three GPCRs while maintaining their biological activity. We also demonstrated a robust method for assessing binding constants for NK1R-NLPs that interact with SP using FCS. This combined approach should be capable of high-throughput screening for active GPCRs produced by cell-free co-expression. In the future, it will be of interest to build upon these studies to explore mechanisms behind G-protein activation and potential receptor uptake in cells. Chromosomal mutagenesis is a critical genetic tool for the study of bacterial systems. Many bacteria cannot be readily transformed with linear DNA fragments, greatly limiting our ability to introduce chromosomal mutations. Recombineering, a method that involves expression of bacteriophage recombination proteins, has transformed our ability to engineer bacterial chromosomes using linear dsDNA or ssDNA. Thus, it is now possible to rapidly introduce point mutations, insertions, gene deletions, and epitope tags into the chromosomes of many bacterial species. Existing recombineering methods involve two key components: expression of bacteriophage recombination proteins, and generation of suitable DNA fragments for recombination. The latter component typically relies on specific DNA templates for PCR-based synthesis of dsDNA. Most described recombineering systems vary only in the DNA templates used, i.e. different selectable markers. Despite the wide variety of recombineering systems now available for enterobacteria such as Escherichia coli and Salmonella enterica, many have important limitations. We have not been able to determine why FRUIT is so much more efficient. Given that the only difference between the two techniques is the marker used for selection, we propose that the choice of marker may have large effects on the efficiency of recombineering. Recombineering using thyA has been described previously for BAC mutagenesis. Hence, our work is an extension of prior studies using this marker. Similarly, other methods have been described that use recombineering substrates with marker genes or cassettes that can be both selected and counter-selected. These include use of tolC and galK as single-gene markers, and tetAR as a two-gene cassette. Cassettes with separate selectable and counter-selectable markers have also been developed, e.g. chloramphenicol resistance gene and sacB, which can be counterselected by growth on media containing sucrose.