Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY DIY-GEM: Tools for citizen-scientists Baltimore-US Baltimore-US DIY-GEM: Tools for citizen-scientists 1 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY A little bit about who we are We are: ◮ Excited by the idea of ubiquitous synthetic biology. ◮ Sympathetic to the DIY ethic. We want to know: ? biology = breadboards electronics = amateur rockets physics and how can we help? Baltimore-US DIY-GEM: Tools for citizen-scientists 2 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY What is the potential of DIY science? Imagine if you could brew polymerase in an afternoon: ◮ PCR in high schools? ◮ External wet drives? ◮ Would you like some genetic sequencing with your birdwatching? Baltimore-US DIY-GEM: Tools for citizen-scientists 3 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY How should we think about this potential? Baltimore-US DIY-GEM: Tools for citizen-scientists 4 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY How should we think about this potential? Baltimore-US DIY-GEM: Tools for citizen-scientists 4 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY How should we think about this potential? ◮ Invited liasons from the WMD Directorate to speak to our group. ◮ Attended Presidential Summit on Bioethics ◮ Participated in 1st FBI-DIYBIO workshop Baltimore-US DIY-GEM: Tools for citizen-scientists 4 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY Our Project: a kit for citizen-scientists We have developed: ◮ BioBricked Taq polymerase ◮ A gel-electrophoresis kit (some assembly required) We are in the process of developing: ◮ PCRIY: an at-home thermal cycler. Baltimore-US DIY-GEM: Tools for citizen-scientists 5 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY Taq Polymerase on the cheap ◮ Polymerase is a necessary ingredient for PCR. ◮ But it’s expensive: on the order of 10 cents / unit of activity. ◮ And what if you can’t find someone to sell it to you? Baltimore-US DIY-GEM: Tools for citizen-scientists 6 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY How we planned to do it 1. Design Taq primers with BioBrick prefix and suffix. 2. PCR Taq, yielding Taq with BioBrick ’fixes attached. 3. Cut and ligate into standard assembly vector. Baltimore-US DIY-GEM: Tools for citizen-scientists 7 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY A bit of a problem Pst I site 3' 5' 3' 5' T. aquaticus Pol I gene Baltimore-US DIY-GEM: Tools for citizen-scientists 8 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY Why is this a problem? P S X Pol1 E P X E Cut with Spe I and Pst I Baltimore-US DIY-GEM: Tools for citizen-scientists 9 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY Remedy: overlap extension mutagenesis Pst I site Pol I gene CT G GAC 5' 3' BB prefix + Pol 1 5' 3' GA A CTG Mutagenic primer PCR Reaction #1 PstI site disrupted BB prefix Pol1 gene CT T GAC 5' 3' 3' 5' Baltimore-US DIY-GEM: Tools for citizen-scientists 10 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY . . . Simile for complement Pol I + BB suffix Mutagenic primer Pol I gene CT T GAC 3' 5' 3' 5' GA C CTG Pst I site PCR Reaction #2 Pst I site disrupted GA A CTG BB suffix 5' 3' 3' 5' Baltimore-US DIY-GEM: Tools for citizen-scientists 11 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY . . . and combine! Product from Reaction #2 Product from Reaction #1 BB prefix Pol I CT T GAC 5' 3' 5' 3' 5' 3' 5' 3' GA A CTG BB suffix Denature, Anneal BB prefix Pol I CT T GAC 5' 3' 3' 5' GA A CTG BB suffix Extend w/ Pfu T urbo BB prefix Pol I CT T GAC 3' 5' 5' 3' GA A CTG BB suffix Baltimore-US DIY-GEM: Tools for citizen-scientists 12 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY Results of PCR amplification of T. aquaticus 5 and 3 regions ◮ Lane 1: 1Kb ladder ◮ Lane 2: 100 bp ladder ◮ Lane 3: PCR using BB prefix and mutagenic primer #1 ◮ Lane 4: PCR using BB suffix and mutagenic primer #2. PCR reactions were performed using T. aquaticus YT-1 as template. Baltimore-US DIY-GEM: Tools for citizen-scientists 13 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY Results of overlap extension ◮ Lane 1: 1 Kb ladder, ◮ Lanes 2-8: Results of temperature/DMSO gradient for annealing temperature. 2.5 Kb band in lane 6 excised and purified using Qiagen Gel Extraction Kit. Baltimore-US DIY-GEM: Tools for citizen-scientists 14 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY Overlap extension disappoints ◮ Initial rounds PCR yielded products of approximately the correct size. ◮ Reaction #1: 1500bp ◮ Reaction #2: 1000bp ◮ Yet we were unable to isolate a correctly-sized product for the overlap (never able to amplify): ◮ Expected: ca. 2500bp ◮ Consistently yielded 900bp instead. ◮ This suggested that the binding sites for our PCR primers were not unique. ◮ With the deadline approaching, we attempted another protocol. Baltimore-US DIY-GEM: Tools for citizen-scientists 15 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY Site-directed Mutagenesis Note: ◮ Overlap extension attempted with genomic Taq DNA. ◮ Quik-change protocol performed with a cloned Pol I gene. Baltimore-US DIY-GEM: Tools for citizen-scientists 16 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY Colony PCR using BB ’fixes on colonies following Quik-change mutagenesis ◮ Lane 1: 1Kb ladder ◮ Lane 2: 100 bp ladder ◮ Lane 3: T. aquaticus genomic DNA (+) control ◮ Lane 4: colony pool A ◮ Lane 5: colony pool B Dpn I digestion and transformation into NEB 10 α ◮ Lane 6: colony pool C cells. Bands were excised from the gel, purified ◮ Lane 7: colony pool D using the Qiagen Gel Extraction Kit, digested ◮ Lane 8: colony pool E with Eco RI-HF and Pst I-HF, gel purified again and ligated into plasmid pSB1C3. Baltimore-US DIY-GEM: Tools for citizen-scientists 17 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY Success! Baltimore-US DIY-GEM: Tools for citizen-scientists 18 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY Future Directions ◮ promoter, RBS, purification tag ◮ validation, assay ◮ purification kit We look forward to developing: ◮ BioBricked restriction enzymes, ligase ◮ A purification kit for Taq ◮ an oligonucleotide synthesizer. Baltimore-US DIY-GEM: Tools for citizen-scientists 19 / 28
Y Y BALTIMORE COMMUNITY COLLEGE OF BALTIMORE COUNTY COMMUNITY COLLEGE OF BALTIMORE COUNTY TOWSON UNIVERSITY TOWSON UNIVERSITY LOYOLA UNIVERSITY LOYOLA UNIVERSITY Hardware What if we had more economical tools? ◮ Wider access ◮ Higher throughput Our goal was to develop hardware with these specific attributes: ◮ Inexpensive ◮ Ease of construction ◮ Ease of use of the final product ◮ Replicates common lab equipment Baltimore-US DIY-GEM: Tools for citizen-scientists 20 / 28
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