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Mo's Barret Presentation


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Mo's Barret Presentation

  1. 1. Bioengineering Red Fluorescent Tags from Thermosynechococcus elongatus <ul><li>0911T Characterization & Contamination </li></ul><ul><li>Mo Kaze </li></ul>
  2. 2. Actual Result
  3. 3. Expected Result
  4. 4. What ’ s the opposite of Eureka? <ul><li>Identify the problem (What happened?) </li></ul><ul><li>Hypothesizing explanations for unexpected result (Why did it happen?) </li></ul><ul><li>Locate source (How did it happen?) </li></ul><ul><li>Find the real 911T ! </li></ul>
  5. 5. Identifying the problem <ul><li>Pelleted proteins were the wrong color therefore, </li></ul><ul><ul><li>Protein expressed was NOT the correct gene </li></ul></ul><ul><li>So which one was it? </li></ul>
  6. 6. Hypothesizing possibilities <ul><li>Exploring potential reasons for results </li></ul><ul><ul><li>Sample information was labeled incorrectly, entirely different gene was expressed </li></ul></ul><ul><ul><li>Samples themselves were contaminated by another plasmid and the blue clone was dominant </li></ul></ul>
  7. 7. Contamination <ul><li>Exploring possible sources of contamination </li></ul><ul><ul><li>Our glycerol stock of E.coli cells (LMG-194-PCB or DH5α) was contaminated with the blue (mutated) clone </li></ul></ul><ul><ul><li>Tools or other implements contaminated our cultures during the expression process and the blue clone was dominant </li></ul></ul>
  8. 8. Procedures: preparing DNA for sequencing <ul><li>Mini prepping DNA </li></ul><ul><ul><li>Grow 2 mL cultures from the blue pellet </li></ul></ul><ul><ul><li>Centrifuge to collect the bacteria </li></ul></ul><ul><ul><li>Add lysis buffer to E.coli </li></ul></ul><ul><ul><li>Extract proteins, chromosomal DNA, cell walls </li></ul></ul><ul><ul><li>Centrifuge to remove debris </li></ul></ul><ul><ul><li>Bind and wash and elute plasmid DNA </li></ul></ul><ul><ul><li>Miniprep product = pure plasmid DNA </li></ul></ul><ul><ul><li>Make 2 separate samples for sequencing </li></ul></ul><ul><ul><ul><li>One with pBAD forward DNA PCR primers </li></ul></ul></ul><ul><ul><ul><li>One with CBD reverse DNA PCR primers </li></ul></ul></ul>
  9. 9. Procedures: Sequencing DNA <ul><li>Off the samples go to UC Berkeley ’ s DNA sequencing facility where their powerful equipment can sequence DNA and have results back in less than 2 days time </li></ul><ul><li> </li></ul>
  10. 10. Procedures: Interpreting DNA results <ul><li>What we get back from UCB: </li></ul><ul><ul><li>Chromatograph </li></ul></ul>
  12. 12. Procedures: Interpreting DNA results <ul><li>Using a free tool provided by UCSD we upload sequences and compare them </li></ul><ul><li>We have the entire gene </li></ul><ul><li>sequences uploaded to the </li></ul><ul><li>database already so we </li></ul><ul><li>can compare our sequence </li></ul><ul><li>to the known, published </li></ul><ul><li>gene sequences from </li></ul><ul><li>T.elongatus </li></ul>
  13. 13. Procedures: comparing sequences <ul><li>Sequenced &quot;911T&quot; aligned with Published 911 </li></ul>
  14. 14. Procedures: comparing sequences <ul><li>Sequenced &quot;911T&quot; aligned with Published 569 </li></ul>
  15. 15. Sequence comparison results <ul><li>This was the first explicit confirmation of what we suspected </li></ul><ul><li>“ 0911T ” was NOT what we had expressed </li></ul><ul><li>0911T was actually 0569TM another one of our T.elongatus genes </li></ul>
  16. 16. Tracking down the source <ul><li>Using lab books to determine possible dates where mislabeling could have occurred </li></ul><ul><li>Locating properly labeled glycerol stocks prior to the suspected date and then sequencing plasmid DNA </li></ul>
  17. 17. Moving forward <ul><li>Ensuring the correct gene is being used </li></ul><ul><ul><li>Double checking DNA sequence data </li></ul></ul><ul><ul><li>Segregating samples </li></ul></ul><ul><ul><li>Using consistent, sterile technique </li></ul></ul><ul><ul><li>Start expression process to verify expected result: </li></ul></ul><ul><ul><li>Yellow! </li></ul></ul>
  18. 18. Thank you Spiller Lab! <ul><li>Many thanks to: Monica, Alex, Kristen, Rosa, Valerie, Stefanie, Ling, and Dr. Susan Spiller </li></ul>