Team:UMaryland/notebook

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<li class="item101 level0 first"><span class="mymarg"><a class="yjanchor  first" href="/iGemWiki/"><span class="yjm_has_none"><span class="yjm_title">Homepage</span></span></a></span></li><li class="item104 level0"><span class="mymarg"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/photos"><span class="yjm_has_none"><span class="yjm_title">Photos</span></span></a></span></li><li class="haschild item105 level0"><span class="child"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/meetourteam"><span class="yjm_has_none"><span class="yjm_title">Team</span></span></a></span><ul class="subul_main level1"><li class="bl"></li><li class="tl"></li><li class="tr"></li><li class="item293 level1 first"><span class="mymarg"><a class="yjanchor  first" href="https://2014.igem.org/Team:UMaryland/meetourteam"><span class="yjm_has_none"><span class="yjm_title">Meet Our Team Members</span></span></a></span></li><li class="item116 level1"><span class="mymarg"><a class="yjanchor " href="https://igem.org/Team.cgi?id=1489"><span class="yjm_has_none"><span class="yjm_title">iGEM Page</span></span></a></span></li><li class="item117 level1"><span class="mymarg"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/team/attributions"><span class="yjm_has_none"><span class="yjm_title">Attributions</span></span></a></span></li><li class="item300 level1 lilast"><span class="mymarg"><a class="yjanchor last" href="https://2014.igem.org/Team:UMaryland/team/sponsors"><span class="yjm_has_none"><span class="yjm_title">Sponsors</span></span></a></span></li><li class="right"></li><li class="br"></li></ul></li><li class="haschild item112 level0"><span class="child"><a class="yjanchor yjscroll" href="https://2014.igem.org/Team:UMaryland/project/overview"><span class="yjm_has_none"><span class="yjm_title">Project</span></span></a></span><ul class="subul_main level1"><li class="bl"></li><li class="tl"></li><li class="tr"></li><li class="item138 level1 first"><span class="mymarg"><a class="yjanchor  first" href="https://2014.igem.org/Team:UMaryland/project/overview"><span class="yjm_has_none"><span class="yjm_title">Overview</span></span></a></span></li><li class="item145 level1"><span class="mymarg"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/project/background"><span class="yjm_has_none"><span class="yjm_title">Background</span></span></a></span></li><li class="item144 level1"><span class="mymarg"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/project/biobricks"><span class="yjm_has_none"><span class="yjm_title">parts (BioBricks)</span></span></a></span></li><li class="item102 level1"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/project/results" target="_blank" ><span class="yjm_has_none"><span class="yjm_title">Results</span></span></a></li><li class="item254 level1"><span class="mymarg"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/project/futureapplications"><span class="yjm_has_none"><span class="yjm_title">future applications</span></span></a></span></li><li class="item301 level1 lilast"><span class="mymarg"><a class="yjanchor last" href="https://igem.org/Safety/Safety_Form?team_id=1489"><span class="yjm_has_none"><span class="yjm_title">Safety</span></span></a></span></li><li class="right"></li><li class="br"></li></ul></li><li class=" active haschild item234 level0"><span class="child"><a class="yjanchor  activepath " href="https://2014.igem.org/Team:UMaryland/notebook"><span class="yjm_has_none"><span class="yjm_title">Notebook</span></span></a></span><ul class="subul_main level1"><li class="bl"></li><li class="tl"></li><li class="tr"></li><li id="current" class=" active item303 level1 first lilast"><span class="mymarg"><a class="yjanchor  firstlast activepath " href="https://2014.igem.org/Team:UMaryland/notebook"><span class="yjm_has_none"><span class="yjm_title">Wetlab Notebook</span></span></a></span></li><li class="right"></li><li class="br"></li></ul></li><li class="haschild item123 level0"><span class="child"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/humanprac/overview"><span class="yjm_has_none"><span class="yjm_title">Human Practices</span></span></a></span><ul class="subul_main level1"><li class="bl"></li><li class="tl"></li><li class="tr"></li><li class="item106 level1 first"><span class="mymarg"><a class="yjanchor  first" href="https://2014.igem.org/Team:UMaryland/humanprac/overview"><span class="yjm_has_none"><span class="yjm_title">Overview | Mission Statement</span></span></a></span></li><li class="haschild item129 level1"><span class="child subparent"><a class="yjanchor  grouptitle" href="https://2014.igem.org/Team:UMaryland/humanprac/publicschool"><span class="yjm_has_none"><span class="yjm_title">Public School Outreach</span></span></a></span><ul class="subul_main level2"><li class="bl"></li><li class="tl"></li><li class="tr"></li><li class="item134 level2 first lilast"><span class="mymarg"><a class="yjanchor  firstlast" href="https://2014.igem.org/Team:UMaryland/humanprac/publicschool"><span class="yjm_has_none"><span class="yjm_title">HighSchool</span></span></a></span></li><li class="right"></li><li class="br"></li></ul></li><li class="item131 level1"><span class="mymarg"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/humanprac/community"><span class="yjm_has_none"><span class="yjm_title">Community Interaction</span></span></a></span></li><li class="item132 level1 lilast"><span class="mymarg"><a class="yjanchor last" href="https://2014.igem.org/Team:UMaryland/humanprac/publicawareness"><span class="yjm_has_none"><span class="yjm_title">Public Awareness</span></span></a></span></li><li class="right"></li><li class="br"></li></ul></li></ul></div>
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<li class="item101 level0 first"><span class="mymarg"><a class="yjanchor  first" href="https://2014.igem.org/Team:UMaryland"><span class="yjm_has_none"><span class="yjm_title">Homepage</span></span></a></span></li><li class="item104 level0"><span class="mymarg"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/photos"><span class="yjm_has_none"><span class="yjm_title">Photos</span></span></a></span></li><li class="haschild item105 level0"><span class="child"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/meetourteam"><span class="yjm_has_none"><span class="yjm_title">Team</span></span></a></span><ul class="subul_main level1"><li class="bl"></li><li class="tl"></li><li class="tr"></li><li class="item293 level1 first"><span class="mymarg"><a class="yjanchor  first" href="https://2014.igem.org/Team:UMaryland/meetourteam"><span class="yjm_has_none"><span class="yjm_title">Meet Our Team Members</span></span></a></span></li><li class="item116 level1"><span class="mymarg"><a class="yjanchor " href="https://igem.org/Team.cgi?id=1489"><span class="yjm_has_none"><span class="yjm_title">iGEM Page</span></span></a></span></li><li class="item117 level1"><span class="mymarg"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/team/attributions"><span class="yjm_has_none"><span class="yjm_title">Attributions</span></span></a></span></li><li class="item300 level1 lilast"><span class="mymarg"><a class="yjanchor last" href="https://2014.igem.org/Team:UMaryland/team/sponsors"><span class="yjm_has_none"><span class="yjm_title">Sponsors</span></span></a></span></li><li class="right"></li><li class="br"></li></ul></li><li class="haschild item112 level0"><span class="child"><a class="yjanchor yjscroll" href="https://2014.igem.org/Team:UMaryland/project/overview"><span class="yjm_has_none"><span class="yjm_title">Project</span></span></a></span><ul class="subul_main level1"><li class="bl"></li><li class="tl"></li><li class="tr"></li><li class="item138 level1 first"><span class="mymarg"><a class="yjanchor  first" href="https://2014.igem.org/Team:UMaryland/project/overview"><span class="yjm_has_none"><span class="yjm_title">Overview</span></span></a></span></li><li class="item145 level1"><span class="mymarg"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/project/background"><span class="yjm_has_none"><span class="yjm_title">Background</span></span></a></span></li><li class="item144 level1"><span class="mymarg"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/project/biobricks"><span class="yjm_has_none"><span class="yjm_title">parts (BioBricks)</span></span></a></span></li><li class="item102 level1"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/project/results" target="_blank" ><span class="yjm_has_none"><span class="yjm_title">Results</span></span></a></li><li class="item254 level1"><span class="mymarg"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/project/futureapplications"><span class="yjm_has_none"><span class="yjm_title">future applications</span></span></a></span></li><li class="item301 level1 lilast"><span class="mymarg"><a class="yjanchor last" href="https://igem.org/Safety/Safety_Form?team_id=1489"><span class="yjm_has_none"><span class="yjm_title">Safety</span></span></a></span></li><li class="right"></li><li class="br"></li></ul></li><li class=" active haschild item234 level0"><span class="child"><a class="yjanchor  activepath " href="https://2014.igem.org/Team:UMaryland/notebook"><span class="yjm_has_none"><span class="yjm_title">Notebook</span></span></a></span><ul class="subul_main level1"><li class="bl"></li><li class="tl"></li><li class="tr"></li><li id="current" class=" active item303 level1 first lilast"><span class="mymarg"><a class="yjanchor  firstlast activepath " href="https://2014.igem.org/Team:UMaryland/notebook"><span class="yjm_has_none"><span class="yjm_title">Wetlab Notebook</span></span></a></span></li><li class="right"></li><li class="br"></li></ul></li><li class="haschild item123 level0"><span class="child"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/humanprac/overview"><span class="yjm_has_none"><span class="yjm_title">Human Practices</span></span></a></span><ul class="subul_main level1"><li class="bl"></li><li class="tl"></li><li class="tr"></li><li class="item106 level1 first"><span class="mymarg"><a class="yjanchor  first" href="https://2014.igem.org/Team:UMaryland/humanprac/overview"><span class="yjm_has_none"><span class="yjm_title">Overview | Mission Statement</span></span></a></span></li><li class="haschild item129 level1"><span class="child subparent"><a class="yjanchor  grouptitle" href="https://2014.igem.org/Team:UMaryland/humanprac/publicschool"><span class="yjm_has_none"><span class="yjm_title">Public School Outreach</span></span></a></span><ul class="subul_main level2"><li class="bl"></li><li class="tl"></li><li class="tr"></li><li class="item134 level2 first lilast"><span class="mymarg"><a class="yjanchor  firstlast" href="https://2014.igem.org/Team:UMaryland/humanprac/publicschool"><span class="yjm_has_none"><span class="yjm_title">HighSchool</span></span></a></span></li><li class="right"></li><li class="br"></li></ul></li><li class="item131 level1"><span class="mymarg"><a class="yjanchor " href="https://2014.igem.org/Team:UMaryland/humanprac/community"><span class="yjm_has_none"><span class="yjm_title">Community Interaction</span></span></a></span></li><li class="item132 level1 lilast"><span class="mymarg"><a class="yjanchor last" href="https://2014.igem.org/Team:UMaryland/humanprac/publicawareness"><span class="yjm_has_none"><span class="yjm_title">Public Awareness</span></span></a></span></li><li class="right"></li><li class="br"></li></ul></li></ul></div>
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<p class="MsoNormal">These are our two main project BioBricks: the Bovine galectin alone in pSB1C3-pBAD-RBS (BBa_K1489000) and the Bovine galectin gene fused to the end of the OmpA gene in the same vector (BBa_K1489004). Additionally, since the pSB1C3 backbone has a SacI site (which pSB1A2 did not) and OmpA has a SacI site (as designed by the creators of that BioBrick), the pSB1C3-OmpA which we created back in June had two unusable SacI sites. In order to maintain the integrity of that BioBrick with its unique restriction sites (along with the standard EcoRI and PstI sites), we also performed SDM on the pSB1C3-OmpA (BBa_K1489002) to replace the now-useless SacI site at the end of the OmpA gene with a KasI site, creating our fourth BioBrick (BBa_K1489003).</p>
<p class="MsoNormal">These are our two main project BioBricks: the Bovine galectin alone in pSB1C3-pBAD-RBS (BBa_K1489000) and the Bovine galectin gene fused to the end of the OmpA gene in the same vector (BBa_K1489004). Additionally, since the pSB1C3 backbone has a SacI site (which pSB1A2 did not) and OmpA has a SacI site (as designed by the creators of that BioBrick), the pSB1C3-OmpA which we created back in June had two unusable SacI sites. In order to maintain the integrity of that BioBrick with its unique restriction sites (along with the standard EcoRI and PstI sites), we also performed SDM on the pSB1C3-OmpA (BBa_K1489002) to replace the now-useless SacI site at the end of the OmpA gene with a KasI site, creating our fourth BioBrick (BBa_K1489003).</p>
<p class="MsoNormal"><strong>October</strong></p>
<p class="MsoNormal"><strong>October</strong></p>
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<p class="MsoNormal">With the BioBricks made, the team began preliminary protein expression tests. Multiple cultures of DH5α E. coli, transformed with either the bovine galectin or OmpA-BvGal1 BioBricks, were grown up to a noticeable OD. They were then induced with arabinose at increasing concentrations, from no induction to 0.2% m/v. After further incubation of the bacteria to allow for protein production, a 1 mL sample of each culture was pelleted. The cells were then lysed by resuspension in 20 μL SDS and boiling the solution, yielding a soluble protein fraction in the supernatant after centrifugation. The remaining pellet was boiled in 100 μL SDS with harsher conditions in order to extract as much insoluble protein as possible. These protein samples (soluble and insoluble fractions) were run through SDS-PAGE, followed by western blotting (taking advantage of the His6 tag placed in the synthesized bovine galectin-1). Anti-mouse was used for the secondary antibody. Expression of the bovine galectin BioBrick (BBa_K1489000) appeared strong and at the correct size, but expression of OmpA-BvGal1 (BBa_K1489004) featured weak bands in both the induced and uninduced samples, though of correct size.</p>
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