Engineering a blue light inducible SpyCatcher system (BLISS) as a tool for protein photopatterning and optogenetics

The SpyTag-SpyCatcher protein conjugation system has recently exploded in popularity due to its fast kinetics and high yield under biologically favorable conditions in both in vitro and intracellular settings. We imagine we can further expand the utility of this system by introducing the ability to spatially and temporally control the conjugation event. Taking inspiration from photoreceptor proteins in nature, we designed a method to integrate light dependency into the protein conjugation reaction. The light-oxygen-voltage 2 domain of Avena sativa (AsLOV2) undergoes a dramatic conformational change in response to blue light. We have thus genetically fused the SpyTag into the AsLOV2 domain to create a Blue Light Inducible SpyCatcher System (BLISS). In this design (Figure 1), the SpyTag is blocked from reacting with the SpyCatcher in the dark, but upon irradiation with blue light, the Jα-helix of the AsLOV2 undocks to expose the SpyTag. We screened several likely insertion points in the Jα-helix, and found a variant with desirable light switching behavior where after one hour of irradiation, the reaction is 80% complete, while only 10% of the AsLOV2-SpyTag protein reacted in the dark. This reaction can be quenched within minutes by returning the reaction to the dark. We demonstrated the spatial aspect of this light control mechanism through photopatterning proteins onto Ni-NTA coated slides. As our system is made solely from protein components, which can be genetically encoded, we can extend the same spatiotemporal control of proteins inside cells. We anticipate BLISS will be a strong tool for fabricating protein microassays, crafting biomaterial composition, as well as optically controlling enzyme activity and protein localization in cells. Please click Additional Files below to see the full abstract

STATE OF THE - ENVIRONMENT - REPORT 2011

The government in the United Kingdom removed the 2007 National Indicator Framework and instated budget cuts to local authorities. Still, Croydon's Sustainable Development Service finds the annual production of the State of the Environment Report important to pursuing the borough's environmental interests. This project proposes a cost efficient process for generating future editions of the Report. The process is consistent with practices of boroughs in England, and includes a workbook to store indicator data and templates to generate the report

Heating and Cooling in a Packed Bed

The purpose of this research was to examine heat transfer in wall-heated and wall-cooled packed beds. In previous research at Worcester Polytechnic Institute, cooling was found to trend higher than heating. Through changes in the computer model, calculated temperature profiles were closer to experimental profiles. When changing the experimental procedure to reduce re-packings, the trends in parameters were less scattered. Using these alterations, heating and cooling were found to be more similar than what was observed in the past

Synergistic Phytochemicals Fail to Protect Against Ovariectomy Induced Bone Loss in Rats

Menopause induces a loss of bone as a result of estrogen deficiency. Despite pharmaceutical options for the treatment of osteopenia and osteoporosis, many aging women use dietary supplements with estrogenic activity to prevent bone loss and other menopausal-related symptoms. Such supplements are yet to be tested for efficacy against a Food and Drug Administration (FDA) approved medication for menopausal bone loss such as zoledronic acid (ZA). The postmenopausal rat model was used to investigate the efficacy of various synergistic phytochemical blends mixed into the diet for 16 weeks. Retired-breeder, Fischer 344 rats were randomly assigned to sham or ovariectomy surgery and 4 treatment groups: ZA; genistein supplementation; and a low dose and high dose blend of genistein, resveratrol, and quercetin. Ovariectomy resulted in a loss of both trabecular and cortical bone which was prevented with ZA. The phytochemical blends tested were unable to reverse these losses. Despite the lack of effectiveness in preventing bone loss, a significant dose-response trend was observed in the phytochemical-rich diets in bone adipocyte number compared to ovariectomized control rats. Data from this study indicate that estrogenic phytochemicals are not as efficacious as ZA in preventing menopausal-related bone loss but may have beneficial effects on bone marrow adiposity in rats