Team:Cornell/Sandbox/notebook

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<div>May</div>
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<h3>March</h3>
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<h3 class="centered">Week 1</h3>
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We began the summer by holding a synthetic biology bootcamp in the DeLisa Lab. The purpose of this bootcamp was both to introduce new members to techniques in molecular biology and to get a running start on the cloning work for our project. During bootcamp, we successfully constructed both versions of our arsenic reporter, and attempted a Gibson assembly of a naphthalene-degrading plasmid.  
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<h5 class="centered">(05/01 - 05/07)</h5>
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In late June, we transitioned from bootcamp to our permanent bench space in Dr. Archer’s lab in Weill Hall. After spending a few weeks setting up the lab space troubleshooting general issues, we successfully constructed both versions of our salicylate reporter and began an alternative approach to construct a plasmid with a naphthalene-degrading (nah) operon. In parallel, we realized that electroporation efficiency for Shewanella transformation is less than optimal—to say the least. However, we were able to conjugate our constructs into Shewanella using a protocol provided by Dr. Gralnick.  
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As we transitioned into the fall semester in late August, wetlab work was divided into subprojects that could be accomplished in parallel. Subproject leaders independently worked on (1) characterizing our engineered Shewanella strains using reactors in the Angenent lab, (2) characterizing inducible promoters via qPCR of mtrB transcript in response to analyte, (3) characterizing inducible promoters in Shewanella via fluorescent reporters,(4) appending His-tags to MtrB in order to perform immunoassays, (5) performing site-directed mutagenesis on the nah operon to delete BioBrick cutsites, (6) constructing our final naphthalene-degrading plasmid to be conjugated into Shewanella, and (7) confirming that strains carrying the naphthalene-degrading plasmid can actually eat naphthalene.
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<div class="entry nb-wet">
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<h6>WetTest</h6>
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Sed elementum accumsan vestibulum. Cras sit amet nulla sapien, malesuada ullamcorper lorem. Proin pharetra justo ut justo rutrum placerat. Nunc posuere mattis ornare. Curabitur in lectus orci. Fusce tempus feugiat malesuada. Vivamus convallis luctus accumsan. Curabitur laoreet ipsum semper sapien vestibulum vitae accumsan felis aliquet. Nam quis interdum enim. Maecenas lacus ante, ultrices a pellentesque eu, vulputate id elit. Nulla blandit nulla sit amet quam vulputate eu scelerisque ligula faucibus. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nullam neque enim, convallis eu tincidunt ut, blandit quis sem.
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<h6>DryTest</h6>
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Sed elementum accumsan vestibulum. Cras sit amet nulla sapien, malesuada ullamcorper lorem. Proin pharetra justo ut justo rutrum placerat. Nunc posuere mattis ornare. Curabitur in lectus orci. Fusce tempus feugiat malesuada. Vivamus convallis luctus accumsan. Curabitur laoreet ipsum semper sapien vestibulum vitae accumsan felis aliquet. Nam quis interdum enim. Maecenas lacus ante, ultrices a pellentesque eu, vulputate id elit. Nulla blandit nulla sit amet quam vulputate eu scelerisque ligula faucibus. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nullam neque enim, convallis eu tincidunt ut, blandit quis sem.
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<h6>ModelTest</h6>
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<h6>HPracTest</h6>
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<h6>WikiTest</h6>
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<h6>AnimTest</h6>
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Integer bibendum venenatis lectus, vitae tempus odio consectetur eget. In at quam eros. Sed tincidunt lorem nec est viverra rutrum quis at sem. Vivamus eu neque porta ante laoreet fermentum. Morbi metus tellus, porta sit amet feugiat in, placerat id nisi. Maecenas felis sapien, viverra a posuere sed, imperdiet volutpat est. Mauris nec accumsan nibh. Integer aliquet porta neque, vitae accumsan magna interdum sed. Morbi gravida sodales eros ac dapibus. Mauris luctus augue a massa lacinia convallis. Maecenas et velit orci. Donec aliquam, urna id dictum dignissim, diam urna pellentesque ligula, dignissim malesuada quam lorem ac eros. Donec euismod vestibulum diam, at ultricies mauris aliquam at. Phasellus venenatis metus quis tellus egestas vitae luctus ipsum fermentum. Proin vehicula sagittis condimentum. Donec vitae mi ipsum, sit amet tincidunt augue.
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<h3 class="centered" >Week 2</h3>
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From June 12th-22nd, the DeLisa lab kindly hosted a synthetic biology ‘bootcamp’ for our team members. Dr. Didi Waraho assisted in the instruction of new team members, while Taylor Stevenson helped more experienced members get acquainted with the Gibson assembly method.  
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<h5 class="centered">(05/08 - 05/015)</h5>
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<div class="entry nb-wiki">
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Didi’s group worked on the construction of our arsenic reporter plasmids (now referred to as p14k and p16k). This work involved PCR steps to append non-BioBrick cutsites (AscI and BamHI) to an existing mtrB BioBrick (BBa_K098994) and an arsenic-sensing region (BBa_J33201) in order to introduce modularity for easy promoter switching. By the end of the bootcamp, we confirmed via Sanger sequencing that both versions of our arsenic reporter had been successfully cloned into DH5a, electrocompetent E. coli cells. The next step would be to get the plasmids into our Shewanella strain lacking mtrB on the chromosome (JG700).  
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<h6>WikiTest</h6>
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Taylor’s group had one goal: To get internal PstI and NotI cutsites out of our 10kb naphthalene-degrading (nah) operon while constructing the final plasmid to be transformed into Shewanella. By using mutagenic PCR primers that would introduce silent mutations to get rid of the non-BioBrick compatible internal cutsites, the group planned on ripping the nah operon apart, so to speak, and putting it back together again and into a pBMT-1 backbone via Gibson assembly. Using the NAH7 plasmid (containing nah operon) fromPseudomonas putida G7 (kindly provided by Dr. Gene Madsen), Taylor’s group completed all PCR steps necessary for Gibson assembly, incubated the PCR products with Gibson master mix, and transformed the Gibson products into DH5a—yielding three transformants. At the end of bootcamp, the group ran supercoiled plasmid (miniprepped from transformants) on a gel—as an initial screen for successful construction of our naphthalene-degrading plasmid. As shown below, the gel showed distinct bands for all three lanes. However, further confirmation was necessary, so the next step was to digest the Gibson products to check for correct fragment lengths, and to submit DNA for Sanger sequencing.
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<div id="week3entry" class="nb-week">
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<div id="May" class="twelve columns">
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<h3>May</h3>
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<h3 class="centered">Week 3</h3>
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We have successfully created working arsenic and salicylate sensing constructs and have shown that they are functional in Shewanella and are continuing to characterization of our arsenic and salicylate constructs. Although the response in our reactors is not as sensitive as we would hope, we have various hypotheses that we hope to test. Furthermore, we are using RT-qPCR, western blots, and fluorescence measurements to quantify transcription and translation of mtrB levels in response to varying concentrations of arsenic and salicylate. We also plan on running time courses to determine when mtrB is expressed in the presence of analyte.
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<h5 class="centered">(05/15 - 05/22)</h5>
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<div class="entry nb-anim">
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We are still continuing work on the nah operon. By using site-directed mutagenesis, we aim to make our nah part BioBrick compatible, and we still need to confirm successful conjugation of nah into our Shewanella strains.
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<h6>AnimTest</h6>
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Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer consequat magna non nisi rutrum adipiscing. Donec luctus pharetra turpis, posuere mollis lorem porta at. Ut vestibulum urna id nibh blandit non vehicula felis facilisis. Maecenas dignissim sapien a ipsum tristique condimentum. Aenean convallis lorem sit amet nibh bibendum sollicitudin. Phasellus euismod vulputate odio. Etiam in vestibulum ante. Nullam volutpat volutpat velit, eget facilisis dui dapibus eu. Sed bibendum consectetur velit, porttitor imperdiet urna aliquam eu. Maecenas non arcu sem. Curabitur molestie consectetur ultrices. In convallis metus at velit vehicula placerat. Fusce varius euismod erat ut fringilla. Vivamus posuere, felis nec congue bibendum, leo mauris convallis nulla, vel vestibulum magna leo sit amet quam.
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Latest revision as of 19:43, 13 June 2013

Notebook

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Week 1

(05/01 - 05/07)

This is a Title

My name is Inigo Montoya

TemplateTest2

Hello

TemplateTest1

Hello

WetTest
Sed elementum accumsan vestibulum. Cras sit amet nulla sapien, malesuada ullamcorper lorem. Proin pharetra justo ut justo rutrum placerat. Nunc posuere mattis ornare. Curabitur in lectus orci. Fusce tempus feugiat malesuada. Vivamus convallis luctus accumsan. Curabitur laoreet ipsum semper sapien vestibulum vitae accumsan felis aliquet. Nam quis interdum enim. Maecenas lacus ante, ultrices a pellentesque eu, vulputate id elit. Nulla blandit nulla sit amet quam vulputate eu scelerisque ligula faucibus. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nullam neque enim, convallis eu tincidunt ut, blandit quis sem.
DryTest
Sed elementum accumsan vestibulum. Cras sit amet nulla sapien, malesuada ullamcorper lorem. Proin pharetra justo ut justo rutrum placerat. Nunc posuere mattis ornare. Curabitur in lectus orci. Fusce tempus feugiat malesuada. Vivamus convallis luctus accumsan. Curabitur laoreet ipsum semper sapien vestibulum vitae accumsan felis aliquet. Nam quis interdum enim. Maecenas lacus ante, ultrices a pellentesque eu, vulputate id elit. Nulla blandit nulla sit amet quam vulputate eu scelerisque ligula faucibus. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Nullam neque enim, convallis eu tincidunt ut, blandit quis sem.
ModelTest
Integer bibendum venenatis lectus, vitae tempus odio consectetur eget. In at quam eros. Sed tincidunt lorem nec est viverra rutrum quis at sem. Vivamus eu neque porta ante laoreet fermentum. Morbi metus tellus, porta sit amet feugiat in, placerat id nisi. Maecenas felis sapien, viverra a posuere sed, imperdiet volutpat est. Mauris nec accumsan nibh. Integer aliquet porta neque, vitae accumsan magna interdum sed. Morbi gravida sodales eros ac dapibus. Mauris luctus augue a massa lacinia convallis. Maecenas et velit orci. Donec aliquam, urna id dictum dignissim, diam urna pellentesque ligula, dignissim malesuada quam lorem ac eros. Donec euismod vestibulum diam, at ultricies mauris aliquam at. Phasellus venenatis metus quis tellus egestas vitae luctus ipsum fermentum. Proin vehicula sagittis condimentum. Donec vitae mi ipsum, sit amet tincidunt augue.
HPracTest
Integer bibendum venenatis lectus, vitae tempus odio consectetur eget. In at quam eros. Sed tincidunt lorem nec est viverra rutrum quis at sem. Vivamus eu neque porta ante laoreet fermentum. Morbi metus tellus, porta sit amet feugiat in, placerat id nisi. Maecenas felis sapien, viverra a posuere sed, imperdiet volutpat est. Mauris nec accumsan nibh. Integer aliquet porta neque, vitae accumsan magna interdum sed. Morbi gravida sodales eros ac dapibus. Mauris luctus augue a massa lacinia convallis. Maecenas et velit orci. Donec aliquam, urna id dictum dignissim, diam urna pellentesque ligula, dignissim malesuada quam lorem ac eros. Donec euismod vestibulum diam, at ultricies mauris aliquam at. Phasellus venenatis metus quis tellus egestas vitae luctus ipsum fermentum. Proin vehicula sagittis condimentum. Donec vitae mi ipsum, sit amet tincidunt augue.
WikiTest
Integer bibendum venenatis lectus, vitae tempus odio consectetur eget. In at quam eros. Sed tincidunt lorem nec est viverra rutrum quis at sem. Vivamus eu neque porta ante laoreet fermentum. Morbi metus tellus, porta sit amet feugiat in, placerat id nisi. Maecenas felis sapien, viverra a posuere sed, imperdiet volutpat est. Mauris nec accumsan nibh. Integer aliquet porta neque, vitae accumsan magna interdum sed. Morbi gravida sodales eros ac dapibus. Mauris luctus augue a massa lacinia convallis. Maecenas et velit orci. Donec aliquam, urna id dictum dignissim, diam urna pellentesque ligula, dignissim malesuada quam lorem ac eros. Donec euismod vestibulum diam, at ultricies mauris aliquam at. Phasellus venenatis metus quis tellus egestas vitae luctus ipsum fermentum. Proin vehicula sagittis condimentum. Donec vitae mi ipsum, sit amet tincidunt augue.
AnimTest
Integer bibendum venenatis lectus, vitae tempus odio consectetur eget. In at quam eros. Sed tincidunt lorem nec est viverra rutrum quis at sem. Vivamus eu neque porta ante laoreet fermentum. Morbi metus tellus, porta sit amet feugiat in, placerat id nisi. Maecenas felis sapien, viverra a posuere sed, imperdiet volutpat est. Mauris nec accumsan nibh. Integer aliquet porta neque, vitae accumsan magna interdum sed. Morbi gravida sodales eros ac dapibus. Mauris luctus augue a massa lacinia convallis. Maecenas et velit orci. Donec aliquam, urna id dictum dignissim, diam urna pellentesque ligula, dignissim malesuada quam lorem ac eros. Donec euismod vestibulum diam, at ultricies mauris aliquam at. Phasellus venenatis metus quis tellus egestas vitae luctus ipsum fermentum. Proin vehicula sagittis condimentum. Donec vitae mi ipsum, sit amet tincidunt augue.

Week 2

(05/08 - 05/015)
WikiTest
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer consequat magna non nisi rutrum adipiscing. Donec luctus pharetra turpis, posuere mollis lorem porta at. Ut vestibulum urna id nibh blandit non vehicula felis facilisis. Maecenas dignissim sapien a ipsum tristique condimentum. Aenean convallis lorem sit amet nibh bibendum sollicitudin. Phasellus euismod vulputate odio. Etiam in vestibulum ante. Nullam volutpat volutpat velit, eget facilisis dui dapibus eu. Sed bibendum consectetur velit, porttitor imperdiet urna aliquam eu. Maecenas non arcu sem. Curabitur molestie consectetur ultrices. In convallis metus at velit vehicula placerat. Fusce varius euismod erat ut fringilla. Vivamus posuere, felis nec congue bibendum, leo mauris convallis nulla, vel vestibulum magna leo sit amet quam.

Week 3

(05/15 - 05/22)
AnimTest
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer consequat magna non nisi rutrum adipiscing. Donec luctus pharetra turpis, posuere mollis lorem porta at. Ut vestibulum urna id nibh blandit non vehicula felis facilisis. Maecenas dignissim sapien a ipsum tristique condimentum. Aenean convallis lorem sit amet nibh bibendum sollicitudin. Phasellus euismod vulputate odio. Etiam in vestibulum ante. Nullam volutpat volutpat velit, eget facilisis dui dapibus eu. Sed bibendum consectetur velit, porttitor imperdiet urna aliquam eu. Maecenas non arcu sem. Curabitur molestie consectetur ultrices. In convallis metus at velit vehicula placerat. Fusce varius euismod erat ut fringilla. Vivamus posuere, felis nec congue bibendum, leo mauris convallis nulla, vel vestibulum magna leo sit amet quam.
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