Bangladesh is facing one of the largest mass poisonings in human history with over 77 million people affected by contaminated water each and every day. Over the last few years, the 33 Buckets team has come together to help fulfill this clean water need through filtration, education, and an innovative distribution system to inspire and empower people in Bangladesh and across the world. To start this process, we are working with the Rahima Hoque Girls' school in the rural area of Raipura, Bangladesh to give girls access to clean water where they spend the most time. Through our assessment trip in May 2012, we were able to acquire technical data, community input, and partnerships necessary to move our project forward. Additionally, we realized that in many cases, including the Rahima Hoque school, water problems are not caused by a lack of technology, but rather a lack of utilization and maintenance long-term. To remedy this, 33 Buckets has identified a local filter to have installed at the school, and has designed a small-scale business focused on selling clean water in bulk to the surrounding community. Our price point and association with the Rahima Hoque Girls' school makes our solution sustainable. Plus, with the success of our first site, we see the potential to scale. We already have five nearby schools interested in working to implement similar water projects, and with over 100,000 schools in Bangladesh, many of which lack access to the right water systems, we have a huge opportunity to impact millions of lives. This thesis project describes our journey through this process. First, an introduction to our work prior to the assessment trip and through the ASU EPICS program is given. Second, we include quantitative and qualitative details regarding our May 2012 assessment trip to the Rahima Hoque school and Dhaka. Third, we recount some of the experiences we were able to participate in following the trip to Bangladesh, including the Dell Social Innovation Challenge. Fourth, we examine the technical filtration methods, business model development, and educational materials that will be used to implement our solution this summer. Finally, we include an Appendix with a variety of social venture competitions and applications that we have submitted over the past two years, in addition to other supplementary materials. These are excellent examples of our diligence and provide unique insight into the growth of our project.

Protein affinity reagents have aptly gained profound importance as capture reagents and

drugs in basic research, biotechnology, diagnostics and therapeutics. However, due to the

cost, labor and time associated with production of antibodies focus has recently changed

towards potential of peptides to act as protein affinity reagents. Affinity peptides are easy

to work with, non-immunogenic, cost effective and amenable to scale up. Even though

researchers have developed several affinity peptides, we are far from compiling library of

peptides that encompasses entire human proteome. My thesis describes high throughput

pipeline that can be used to develop and characterize affinity peptides that bind several

discrete sites on target proteins.

Chapter 2 describes optimization of cell-free protein expression using commercially

available translation systems and well-known leader sequences. Presence of internal

ribosome entry site upstream of coding region allows maximal expression in HeLa cell

lysate whereas translation enhancing elements are best suited for expression in rabbit

reticulocyte lysate and wheat germ extract. Use of optimal vector and cell lysate

combination ensures maximum protein expression of DNA libraries.

Chapter 3 describes mRNA display selection methodology for developing affinity peptides

for target proteins using large diversity DNA libraries. I demonstrate that mild denaturant

is not sufficient to increase selection pressure for up to three rounds of selection and

increasing number of selection rounds increases probability of finding affinity peptide s.

These studies enhance fundamental understanding of mRNA display and pave the way

for future optimizations to accelerate convergence of in vitro selections.

Chapter 4 describes a high throughput double membrane dot blot system to rapidly

screen, identify and characterize affinity peptides obtained from selection output. I used

dot blot to screen potential affinity peptides from large diversity of previously

ii

uncharacterized mRNA display selection output. Further characterization of potential

peptides allowed determination of several high affinity peptides from having Kd range 150-

450 nM. Double membrane dot blot is automation amenable, easy and affordable solution

for analyzing selection output and characterizing peptides without ne ed for much

instrumentation.

Together these projects serve as guideline for evolution of cost effective high throughput

pipeline for identification and characterization of affinity peptides.