Team:GeorgiaTech/WeekTen

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<title>10/3-10/9</title><style type="text/css">ol{margin:0;padding:0}p{margin:0}.c0{color:#ffffff;font-size:10pt;text-decoration:underline;font-family:Arial}.c8{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:72.0pt}.c6{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:36.0pt}.c10{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:108.0pt}.c2{color:#ffffff;font-size:10pt;font-family:Arial}.c9{line-height:1.15;text-indent:36.0pt;direction:ltr}.c4{line-height:1.15;text-indent:0pt;direction:ltr}.c15{margin-left:72.0pt}.c7{text-align:center}.c16{list-style-type:lower-latin}.c1{list-style-type:disc}.c19{background-color:#ffffff}.c17{list-style-type:lower-roman}.c12{list-style-type:square}.c11{font-style:italic}.c14{list-style-type:decimal}.c3{font-weight:bold}.c18{margin-left:108.0pt}.c13{margin-left:36.0pt}.c20{border-collapse:collapse}.c5{list-style-type:circle}</style></head><body class="c19"><p class="c4"><span class="c2 c3">10/3/10</span></p><p class="c4"><span class="c0">For Today:</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Run on a gel</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">aoxa, aoxb next to the troubleshooting pcrs from today, specifically of the PCRS using Ompa-F and Aoxa/b-R since they are similar sizes</span></li></ol><ol class="c1"><li class="c6" value="2"><span class="c2">Take out plates from incubator. Record observations</span></li></ol><ol class=""><li class="c8" value="1"><span class="c2">Run colony PCRs on at least 20 colonies per construct (yes 20)</span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">Check which primers to use for colony pcr! We can likely use the primers we already have</span></li><li class="c10"><span class="c2">Run results on gel</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Results: plated colonies from 10/2/10 (Mitesh &amp; Rob):</span></p><p class="c4"><span class="c2">Observed that HybB-ompA-Aox1(a/b) constructs were successfully transformed- strong colony growth and red colonies (strange- given that these were supposedly constructs without RFP). The color strongly suggests RFP. Red colony patterns show no consistent trends- some are densely red in the middle of the plate, others mainly around the edges- red intensity varied widely even on the same plate. </span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4 c7"><img height="268.0" src="http://2010.igem.org/wiki/images/2/28/10-3a.png" width="203.0"></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Colony PCR of HybB-ompA-Aox constructs</span></p><p class="c4"><span class="c2">Picked colonies from plates of HybB-ompA-Aox-RFP and added to 25 ul of water each,10 colonies from Aox1a containing plates, and 10 from Aox1b containing plates:</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">HybB-ompA-Aox1a-RFP: 1.5 mL Tubes 1-10</span></p><p class="c4"><span class="c2">HybB-ompA-Aox1b-RFP: 1.5 mL Tubes 11-20 </span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Used 5 ul for PCR, and added 250 ul of LB media to the rest. </span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Colony PCRs (for each colony): (Rob, Mitesh &amp; Scott)</span></p><p class="c4"><span class="c2">5 uL of cells</span></p><p class="c4"><span class="c2">25.5 uL H2O</span></p><p class="c4"><span class="c2">10 uL GOTAQ</span><span class="c2 c3"> 5X Reaction buffer</span></p><p class="c4"><span class="c2">5 uL HybB-F forward primer</span></p><p class="c4"><span class="c2">5 uL RFP-R reverse primer</span></p><p class="c4"><span class="c2">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c4"><span class="c2">0.5 uL polymerase enzyme, TAQ</span></p><p class="c4"><span class="c2 c3">Total Volume= 50 uL</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Colony PCRs (for each colony):</span></p><p class="c4"><span class="c2">5 uL of cells</span></p><p class="c4"><span class="c2">25.5 uL H2O</span></p><p class="c4"><span class="c2">10 uL GOTAQ</span><span class="c2 c3"> 5X Reaction buffer</span></p><p class="c4"><span class="c2">5 uL HybB-F forward primer</span></p><p class="c4"><span class="c2">5 uL Aox1a or b-R reverse primer</span></p><p class="c4"><span class="c2">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c4"><span class="c2">0.5 uL polymerase enzyme, TAQ</span></p><p class="c4"><span class="c2 c3">Total Volume= 50 uL</span></p><p class="c4"><span class="c2 c3"> </span></p><p class="c4"><span class="c2">Important: For the PCR tubes, the ones labeled 1,3,5,7,9 are treated with Aox1a reverse primer.</span></p><p class="c4"><span class="c2">All the even number (2,4,6,...16,18,20) labeled have RFP-R reverse primer in them. And the tubes labeled 11,13,15,17,19 have Aox 1bR reverse primer in them.</span></p><p class="c4"><span class="c2">PCR start time: 6 PM</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">For Monday:</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Check colony pcr on a gel</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">calculate the predicted sizes and compare to gel results</span></li></ol><ol class="c1"><li class="c6" value="2"><span class="c2">Continue with colony PCRS if more info is required</span></li><li class="c6"><span class="c2">Check triple ligations on gel</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">10/4/2010</span></p><p class="c4"><span class="c0">Protocols</span></p><p class="c4"><span class="c0">&nbsp;</span></p><p class="c4"><span class="c0">1 % Gel to Check colony PCRs from 10/3/2010</span></p><p class="c4"><span class="c2">Started at 9 am</span></p><p class="c4"><span class="c2">Order:ladder:1-6|ladder|7-12|ladder|13-14</span></p><p class="c4"><span class="c2">Important: For the PCR tubes, the ones labeled 1,3,5,7,9 are treated with Aox1a reverse primer.</span></p><p class="c4"><span class="c2">All the even number (2,4,6,...16,18,20) labeled have RFP-R reverse primer in them. And the tubes labeled 11,13,15,17,19 have Aox 1bR reverse primer in them.</span></p><p class="c4"><span class="c2">PCR start time: 6 PM</span></p><p class="c4"><span class="c2">gel start at 10:34 am</span></p><p class="c4"><img height="329.0" src="http://2010.igem.org/wiki/images/a/a0/10-3c.png" width="440.0"></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Predicted Bands:</span></p><p class="c4"><span class="c2">Hyb-F + RFP-R approx 2100-2200 &nbsp;bp</span></p><p class="c4"><span class="c2">Hyb-F + Aoxa-R - approx 1500</span></p><p class="c4"><span class="c2">Hyb-F + Aoxb-R approx 1500</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Observations- 2200 bp band is seen, but other bands are also observed for the Hyb-F and RFP-R reactions</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Suggestions:</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Troubleshoot by PCR sequencing the basic products (e.g. sequence just HybB in one tube, in the next, just OmpA; in the next, just AOX; in the next, just RFP). This will tell us whether each gene is actually present in the cells PCRed.</span></li><li class="c6"><span class="c2">Start over again entirely, from the very beginning, digesting each individual gene, ligating, transforming.</span></li><li class="c6"><span class="c2">Start using controls. When possible, include positive and negative controls. </span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">For example, when doing a double-ligation, for instance, perform experiment alongside designed to fail (e.g. lacking one of the ligation genes) for a negative control. </span></li><li class="c8"><span class="c2">To see if the plasmid pSB1A3 is causing our problems, transformed this plasmid alone directly into E. coli and plate.</span></li></ol><ol class="c1"><li class="c6" value="4"><span class="c2">If pSB1A3 turns out to be the problem, can attempt to use a different plasmid backbone. We&rsquo;ve researched and found plasmid pSB1C3 to have the desired characteristics, including lack of a &nbsp;promoter, beginning EcoRI and ending SpeI restriction sites.</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Plan to go forward:</span></p><p class="c4"><span class="c2">1. Transform &ldquo;plain&rdquo; plasmid pSB1A3 into E. coli today 4 OCT 2010. Check for RFP expression.</span></p><p class="c4"><span class="c2">2. In tandem with step 3, troubleshoot by sequencing the basic gene products in the &ldquo;bad&rdquo; HybB-OmpA-AOX contsructs, looking specifically for each gene.</span></p><p class="c4"><span class="c2">3. In tandem with step 2, re-do the entire construction process from the individual genes up.</span></p><p class="c4"><span class="c2">4. </span><span class="c2 c3">Transform cells using new biobrick vectors, like psb1c3, and use those instead of psb1a3!</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">NOTE: Need to make more media plates!</span></p><p class="c4"><span class="c2 c3">&nbsp;</span></p><p class="c4"><span class="c0">Protocols</span></p><p class="c4"><span class="c0">&nbsp;</span></p><p class="c4"><span class="c0">Transformation of NB cells using pSB1a3</span></p><p class="c4"><span class="c2">10 &#1405;L Nova Blue cells + 5 &#1405;L of pSB1A3 from MiniPrep</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">1. Left cells and plasmid on ice.</span></p><p class="c4"><span class="c2">2. Added plasmid (5&#1405;L) to cells. Mix gently by swirling pipette tip in mixture (DO NOT ASPIRATE).</span></p><p class="c4"><span class="c2">3. Left cells on ice for 30 min.</span></p><p class="c4"><span class="c2">4. Applied heat shock of 45 seconds in 42C bath.</span></p><p class="c4"><span class="c2">5. Put tubes on ice for 2 min.</span></p><p class="c4"><span class="c2">6. Added 250 &#1405;L of LB (room temp.)</span></p><p class="c4"><span class="c2">7. Incubated 1 hour at 37 C</span></p><p class="c4"><span class="c2">8. Plated 100&#1405;L (x2 plates) and left plates in the 37 degrees incubator &nbsp;</span></p><p class="c4"><span class="c2">9. Incubated overnight at 37C. </span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Troubleshooting PCRs</span></p><p class="c4"><span class="c0">For each test (e.g. HybB, OmpA, AOX1a, etc.)</span></p><p class="c4"><span class="c2">2 uL of cells</span></p><p class="c4"><span class="c2">9.4 uL H2O</span></p><p class="c4"><span class="c2">4 uL GOTAQ</span><span class="c2 c3"> 5X Reaction buffer</span></p><p class="c4"><span class="c2">2 uL forward primer</span></p><p class="c4"><span class="c2">2 uL reverse primer</span></p><p class="c4"><span class="c2">0.4 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c4"><span class="c2">0.2 uL polymerase enzyme, GOTAQ</span></p><p class="c4"><span class="c2 c3">Total Volume= 20 uL &nbsp;</span></p><p class="c4"><span class="c2 c3">&nbsp;</span></p><p class="c4"><span class="c0">Combinations</span></p><p class="c4"><span class="c2 c3">A</span><span class="c2">: HybB-OmpA-AOX1a-pSB1A3 in NovaBlue cells, ligated 2 OCT 2010 (Margo + Scott)</span></p><p class="c9"><span class="c2">Selected 1 </span><span class="c2 c3">red</span><span class="c2"> colony, suspended in 20 uL sterile dH2O</span></p><p class="c9"><span class="c2">1: HybB forward &amp; reverse primers</span></p><p class="c9"><span class="c2">2: OmpA forward &amp; reverse primers</span></p><p class="c9"><span class="c2">3: AOX1a forward &amp; reverse primers</span></p><p class="c9"><span class="c2">4: RFP forward &amp; reverse primers</span></p><p class="c9"><span class="c2">&nbsp;</span></p><p class="c9"><span class="c2">Selected 1 </span><span class="c2 c3">white</span><span class="c2"> colony, suspended in 20 uL sterile dH2O</span></p><p class="c9"><span class="c2">5: HybB forward &amp; reverse primers</span></p><p class="c9"><span class="c2">6: OmpA forward &amp; reverse primers</span></p><p class="c9"><span class="c2">7: AOX1a forward &amp; reverse primers</span></p><p class="c9"><span class="c2">8: RFP forward &amp; reverse primers</span></p><p class="c9"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">B</span><span class="c2">: HybB-OmpA-AOX1b-pSB1A3 in NovaBlue cells, ligated 2 OCT 2010 (Margo + Scott)</span></p><p class="c9"><span class="c2">Selected 1 </span><span class="c2 c3">red</span><span class="c2"> colony, suspended in 20 uL sterile dH2O</span></p><p class="c9"><span class="c2">1: HybB forward &amp; reverse primers</span></p><p class="c9"><span class="c2">2: OmpA forward &amp; reverse primers</span></p><p class="c9"><span class="c2">3: AOX1a forward &amp; reverse primers</span></p><p class="c9"><span class="c2">4: RFP forward &amp; reverse primers</span></p><p class="c9"><span class="c2">&nbsp;</span></p><p class="c9"><span class="c2">Selected 1 </span><span class="c2 c3">white</span><span class="c2"> colony, suspended in 20 uL sterile dH2O</span></p><p class="c9"><span class="c2">5: HybB forward &amp; reverse primers</span></p><p class="c9"><span class="c2">6: OmpA forward &amp; reverse primers</span></p><p class="c9"><span class="c2">7: AOX1a forward &amp; reverse primers</span></p><p class="c9"><span class="c2">8: RFP forward &amp; reverse primers</span></p><p class="c4"><span class="c2 c3">&nbsp;</span></p><p class="c4"><span class="c2 c3">P</span><span class="c2">: &ldquo;plain&rdquo; mini-prepped pSB1A3 plasmid</span></p><p class="c4"><span class="c2">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;RFP forward &amp; reverse primers</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Ran a PCR on the samples using 1KB ladder -- ran A 1-7</span></p><p class="c4"><span class="c2">Lane &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1: ladder</span></p><p class="c9"><span class="c2">2: A1</span></p><p class="c9"><span class="c2">3: A2</span></p><p class="c9"><span class="c2">4: A3</span></p><p class="c9"><span class="c2">5: A4</span></p><p class="c9"><span class="c2">6: A5</span></p><p class="c9"><span class="c2">7: A6</span></p><p class="c9"><span class="c2">8: A7</span></p><p class="c9"><span class="c2">&nbsp;</span></p><p class="c4"><img height="297.0" src="http://2010.igem.org/wiki/images/5/57/10-3b.png" width="392.0"></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">Note: need to make a big batch of 1% gels tomorrow since we are running so many samples.</span></p><p class="c4"><span class="c2 c3">Need more agarose from Gaucher Lab. COMPLETED</span></p><p class="c4"><span class="c2 c3">Need more EtBr. COMPLETED</span></p><p class="c4"><span class="c2 c3">Need a 100bp Ladder to see some of this stuff (small constructs) -- we might need to get Ryan to order some. </span></p><p class="c4"><span class="c2 c3">&nbsp;</span></p><p class="c4"><span class="c2 c3">10-5-10</span></p><p class="c4"><span class="c2">Obtain some more agarose and EtBr from Gaucher lab.</span></p><p class="c4"><span class="c2">Make 1% gels. </span></p><p class="c4"><span class="c2 c11 c3">See Protocols page for Preparing 1% Agarose Gels</span></p><p class="c4"><span class="c2">Ran a gel on the remaining PCR samples from 10-4-10</span></p><p class="c4"><span class="c2">Following 1 KB ladder, lanes should read in order:</span></p><p class="c4"><span class="c2">A8, P, B1, B2, B3, B4, B5, B6, B7, B8. </span></p><p class="c4"><span class="c2">No digital picture available, but one was printed and is in the lab, labelled with the date. </span></p><p class="c4"><span class="c2">&nbsp;</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Starting construct building strategy over again</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">Reactions:</span></li></ol><ol class="c14"><li class="c6" value="1"><span class="c2">HyBb F,R (template biobrick)</span></li><li class="c6"><span class="c2">OmpA F,R (template ompa vector)</span></li><li class="c6"><span class="c2">Aox1a F,R (template synthesized gene)</span></li><li class="c6"><span class="c2">Aox1a F,R2 (template synthesized gene)</span></li><li class="c6"><span class="c2">Aox1b F,R (template synthesized gene)</span></li><li class="c6"><span class="c2">Aox1b F,R2 (template synthesized gene)</span></li><li class="c6"><span class="c2">RFP F,R (template RFP plasmid) (from 9.27.2010 miniprep)</span></li><li class="c6"><span class="c2">RFP F2,R &nbsp;(template RFP plasmid) (from 9.27.2010 miniprep)</span></li></ol><p class="c4 c13"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">PCR Protocol (Scott)</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">26.5 uL H2O</span></li><li class="c6"><span class="c2">10 uL </span><span class="c2 c3">PHUSION 5X Reaction buffer</span></li><li class="c6"><span class="c2">5 uL forward primer</span></li><li class="c6"><span class="c2">5 uL reverse primer</span></li><li class="c6"><span class="c2">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></li><li class="c6"><span class="c2 c3">2 uL</span><span class="c2"> template DNA</span></li><li class="c6"><span class="c2">0.5 uL polymerase enzyme, </span><span class="c2 c3">PHUSION</span></li></ol><p class="c4"><span class="c2 c3">Total Volume= 50 uL</span></p><p class="c4"><span class="c2">started 4:33pm</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">10/6/2010</span></p><p class="c4"><span class="c0">Goals</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">The gel from 10/5/2010 of the troubleshooting pcrs, specifically the reaction of pSB1A3 with RFP-F and R, suggests that the vector used for ligation has RFP. We will use anothe rvector from biobrick</span></li><li class="c6"><span class="c2">We want to transform cells with the new biobrick vector today, miniprep the cells tomorrow morning, digest, and ligate to our hyb.ompa+aox that we have or are making now. Finish by friday is the goal. </span></li><li class="c6"><span class="c2">Sequence hyb-RFP (for bronze medal requirements)</span></li><li class="c6"><span class="c2">begin hyb-ompa-rfp building when primers come in</span></li><li class="c6"><span class="c2">I saw Richard&rsquo;s sequence was for psb1c3, but we are using psb1a3; this may a primer and digests! Investigate!</span></li><li class="c6"><span class="c2">Sequence white colonies from the plates on 10/3/2010</span></li><li class="c6"><span class="c2">For today:</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">Reconstitute new biobrick vector (psb1A7) COMPLETED</span></li><li class="c8"><span class="c2">Transform NB cells using new biobrick vector COMPLETED</span></li><li class="c8"><span class="c2">Check PCRs from 10/5/2010 on gel (tubes in yellow pcr box labeled 1-8 and dated 10.5) COMPLETED</span></li><li class="c8"><span class="c2">Make Amp plates</span></li><li class="c8"><span class="c2">Autoclave pipette tips</span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">This involves testing which tips are compatible with ours (we have lots of random tips)</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Notes on Vector-</span></p><p class="c4"><span class="c2">Here is an experience from another team concerning transcription of a product during an &ldquo;off state&rdquo;:</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Our team (</span><span class="c0"><a href="http://www.google.com/url?q=http%3A%2F%2Fparts.mit.edu%2Fwiki%2Findex.php%2FDavidson_2006&amp;sa=D&amp;sntz=1&amp;usg=AFQjCNG5h0TpmlIOGm0emKQ96Nz1nWxszw">Davidson College</a></span><span class="c2">) has used this vector in </span><span class="c2 c11">E. coli</span><span class="c2"> strain JM109 extensively. Clones are Amp resistant and are recovered in reasonable yields from mini preps. One drawback is that the vector has read-through transcription that comes into the cloned BioBrick part from both directions. This problem is a major one when an &quot;off state&quot; is critical for the control of a device. Parts without a promoter are expressed in this vector, regardless of orientation (see</span><span class="c2"><a href="http://www.google.com/url?q=http%3A%2F%2Fpartsregistry.org%2Fwiki%2Findex.php%2FPart%3ABBa_J31009%3ADesign&amp;sa=D&amp;sntz=1&amp;usg=AFQjCNEbgpV7QGpUM3sHpHi6sMXb_tKAoA"> </a></span><span class="c0"><a href="http://www.google.com/url?q=http%3A%2F%2Fpartsregistry.org%2Fwiki%2Findex.php%2FPart%3ABBa_J31009%3ADesign&amp;sa=D&amp;sntz=1&amp;usg=AFQjCNEbgpV7QGpUM3sHpHi6sMXb_tKAoA">pSB1A7 Part Design</a></span><span class="c2"> for details). Reverse read-through might be from the Amp resistance gene. Forward read-through might be from a cryptic promoter, since no known genes in the backbone are in the plus orientation.</span></p><p class="c4"><span class="c2">--</span><span class="c0"><a href="http://www.google.com/url?q=http%3A%2F%2Fpartsregistry.org%2FUser%3AKahaynes&amp;sa=D&amp;sntz=1&amp;usg=AFQjCNGORq4D7Hq4fMg0TPGjwS-hTXsGSQ">Kahaynes</a></span><span class="c2"> 15:48, 23 October 2006 (EDT)</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Alternative vectors:</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">pSB1A7 (2010 Kit plate 3, 15G well, name BBa_K126000)</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">Reasons:</span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">There are double terminators on either side of the multiple cloning site that block any read through or unwanted transcription from the plasmid promoters (plasmid promoters are involved in antibiotic production) </span></li><li class="c10"><span class="c2">Ampicillin resistance</span></li><li class="c10"><span class="c2">Already in kit</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><table cellpadding="0" cellspacing="0" class="c20"><tbody></tbody></table><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Protocols</span></p><p class="c4"><span class="c2">1. Resuspend </span><span class="c2 c3">psb1A7</span><span class="c2"> DNA in 10uL of autoclaved water. </span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4 c13"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">3. Run gels of PCR&rsquo;s on 10-5-10. </span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><img height="330.0" src="http://2010.igem.org/wiki/images/2/29/10-3d.png" width="441.0"></p><p class="c4"><span class="c2">Order:</span></p><ol class="c14"><li class="c6" value="1"><span class="c2">HyBb F,R (template biobrick)</span></li><li class="c6"><span class="c2">OmpA F,R (template ompa vector)</span></li><li class="c6"><span class="c2">Aox1a F,R (template synthesized gene)</span></li><li class="c6"><span class="c2">Aox1a F,R2 (template synthesized gene)</span></li><li class="c6"><span class="c2">Aox1b F,R (template synthesized gene)</span></li><li class="c6"><span class="c2">Aox1b F,R2 (template synthesized gene)</span></li><li class="c6"><span class="c2">RFP F,R (template RFP plasmid) (from 9.27.2010 miniprep)</span></li><li class="c6"><span class="c2">RFP F2,R &nbsp;(template RFP plasmid) (from 9.27.2010 miniprep)</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Growing colony of pSB1a3 transformed cells (Scott)</span></p><p class="c4"><span class="c2">I picked 3 colonies from the plate marked &ldquo;MFC 9.15.2010 C1 &ldquo; from 9.15.2010, with the psb1a3 transformed cells that are slighty pink. I did 3 mL + 3 uL of Carb, and put them in the incubator. </span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">PCR of pSB1A3 (direct from well aliquot) using RFP-F,R</span></p><ol class="c1"><li class="c6" value="8"><span class="c2">27.5 uL H2O</span></li><li class="c6"><span class="c2">10 uL </span><span class="c2 c3">GoTaq 5X Reaction buffer</span></li><li class="c6"><span class="c2">5 uL forward primer (RFP-F) </span></li><li class="c6"><span class="c2">5 uL reverse primer (RFP-R)</span></li><li class="c6"><span class="c2">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></li><li class="c6"><span class="c2 c3">1 uL</span><span class="c2"> template DNA</span></li><li class="c6"><span class="c2">0.5 uL polymerase enzyme, </span><span class="c2 c3">GoTaq</span></li></ol><p class="c4"><span class="c2 c3">Total Volume= 50 uL</span></p><p class="c4"><span class="c2"> Left in block B- Thursday people need to retieve it- Need to gel purify- scroll down to see a lit of things to do for Thursday. These steps are also highligted in blue below. </span></p><p class="c4"><span class="c0">PCR of mRFP miniprep (direct from well aliquot) using RFP-F3,R</span></p><ol class="c1"><li class="c6" value="15"><span class="c2">26.5 uL H2O</span></li><li class="c6"><span class="c2">10 uL </span><span class="c2 c3">GoTaq 5X Reaction buffer</span></li><li class="c6"><span class="c2">5 uL forward primer (RFP-F3) </span></li><li class="c6"><span class="c2">5 uL reverse primer (RFP-R)</span></li><li class="c6"><span class="c2">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></li><li class="c6"><span class="c2 c3">2 uL</span><span class="c2"> template DNA</span></li><li class="c6"><span class="c2">0.5 uL polymerase enzyme, </span><span class="c2 c3">GoTaq</span></li></ol><p class="c4"><span class="c2 c3">Total Volume= 50 uL</span></p><p class="c4"><span class="c2">Left in block B- Thursday people need to retieve it &nbsp;- Scott knows what to do with this. I don&rsquo;t. -Debika</span></p><p class="c4"><span class="c0">&nbsp;</span></p><p class="c4"><span class="c0">&nbsp;</span></p><p class="c4"><span class="c0">Transformation of NB using pSB1A3 direct from Gel aliquot) -</span><span class="c0 c3">completed (Christina, Debika)</span></p><p class="c4"><span class="c2">10 &#1405;L Nova Blue cells + 5 &#1405;L of pSB1A3 from MiniPrep</span></p><p class="c4"><span class="c2"> &nbsp;</span></p><p class="c4"><span class="c2">1. Left cells and plasmid on ice.(Scott)</span></p><p class="c4"><span class="c2">2. Added plasmid (5&#1405;L) to cells. Mix gently by swirling pipette tip in mixture (DO NOT ASPIRATE).</span></p><p class="c4"><span class="c2">3. Left cells on ice for 30 min.</span></p><p class="c4"><span class="c2">4. Applied heat shock of 45 seconds in 42C bath. (Debika)</span></p><p class="c4"><span class="c2">5. Put tubes on ice for 2 min. (I messed up and put in LB in a minute instead of 2 minutes. Left it in ice again for a minute after adding LB). Don&rsquo;t know how that will affect our results. Sorry if this transformation doesn&rsquo;t work). </span></p><p class="c4"><span class="c2">6. Added 250 &#1405;L of LB (room temp.)</span></p><p class="c4"><span class="c2">7. Incubated 1 hour at 37 C</span></p><p class="c4"><span class="c2">8. Plated 100&#1405;L (x2 plates) and left plates in the 37 degrees incubator. (Christina) &nbsp;</span></p><p class="c4"><span class="c2">9. Incubated overnight at 37C. &nbsp;</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Transformation of NB using pSB1A7 direct from Gel aliquot) -</span><span class="c0 c3">completed (Christina)</span></p><p class="c4"><span class="c2">10 &#1405;L Nova Blue cells + 5 &#1405;L of pSB1A3 from MiniPrep</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Add 1uL of resuspended</span><span class="c2 c3"> psb1A7</span><span class="c2"> in 10uL of competent cells (Novablue). (thawed on ice)</span></li></ol><p class="c4"><span class="c2 c11 c3">See Protocols page for Heat Shock Transformation </span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Note: The incubation step was skipped, which likely will lead to little growth</span></p><hr><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">Meeting Notes</span></p><p class="c4"><span class="c0 c3">Priority List</span></p><p class="c9"><span class="c2 c11">Critical</span></p><ol class="c16"><li class="c8" value="1"><span class="c2">hyb.ompa.aox construct (in pSB1A7)</span></li><li class="c8"><span class="c2">hyb.ompa.RFP construct (in pSB1A7) (Scott)</span></li><li class="c8"><span class="c2">Sequencing of constructs and conformation with standard</span></li><li class="c8"><span class="c2">Biobrick</span></li></ol><p class="c4 c13"><span class="c2 c11">Need </span></p><ol class="c16"><li class="c8" value="1"><span class="c2">Calorimetric/Heat &nbsp;(Rob, Christian)</span></li><li class="c8"><span class="c2">Periplasmic Expression Picture (Fluorescent Microscope) (Scott)</span></li><li class="c8"><span class="c2">Cold Shock Quantification (hybB)</span></li><li class="c8"><span class="c2">Modeling (Gita, Mitesh)</span></li></ol><ol class="c17"><li class="c10" value="1"><span class="c2">Liquid culture</span></li><li class="c10"><span class="c2">Solid Media</span></li></ol><p class="c4"><span class="c2">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c2 c11">Want</span></p><ol class="c16"><li class="c8" value="1"><span class="c2">Confirm pSB1A3 has RFP in it</span></li></ol><ol class="c17"><li class="c10" value="1"><span class="c2">Pick colony from plates, liquid culture. (COMPLETED)(SCOTT) &nbsp;&ldquo;MFC 9.15.2010 C1 &ldquo;</span></li></ol><p class="c4 c18"><span class="c2">Thursday: Mini-prep, off to sequencing. Ask Ryan or Megan</span></p><p class="c4 c15"><span class="c2 c11"> &nbsp; &nbsp; ii &nbsp; &nbsp; </span><span class="c2">Aliquot from well, PCR (COMPLETED, DEBIKA, left in right side block overnight)</span></p><p class="c4 c18"><span class="c2">Thursday: gel purify, off to sequencing. Ask Ryan or Megan</span></p><hr><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">Detailed Plan</span></p><p class="c4"><span class="c0 c3">Today (wed)</span></p><p class="c4"><span class="c2 c11">Critical Actions</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">hyb.ompa.rfp construct</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">Start PCR of RFP using new primer using a plasmid with ryan&rsquo;s rfp (pcold and rfp, or the gaucher lab rfp plasmid) (Debika, completed) </span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c11">Want Actions</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">confirm PSB1A3 has RFP</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">Scott</span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">Pick colony from original psb1a3 plates &nbsp;&ldquo;MFC 9.15.2010 C1 &ldquo; and put in liq culture (completed)</span></li></ol><ol class=""><li class="c8" value="2"><span class="c2">Debika</span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">Start PCR of psb1a3 directly from well, using primers RFP-F and RFP-R (Debika, completed)</span></li></ol><ol class=""><li class="c8" value="3"><span class="c2">Christina, Debika,</span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">transformation of NB cells using original psb1a3 aliquot (completed)</span></li></ol><p class="c4 c15"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c11">Other Actions</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">order psb1a7 sequencing primers (the forward psb1a3-F primer is ok, but we need a reverse primer)</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0 c3">Thursday</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">perform a heat shock transformation of psb1a7 -- this could not be done on Wed. because no plates were left to perform this. Had to make plates first.</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">Add 1uL of resuspended</span><span class="c2 c3"> psb1A7</span><span class="c2"> in 10uL of competent cells (Novablue). (thawed on ice)</span></li></ol><p class="c4 c13"><span class="c2 c11 c3">See Protocols page for Heat Shock Transformation </span></p><p class="c9"><span class="c2">&nbsp;</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Mini-prep of psB1A3, send sequence w/ RFP F,R</span></li><li class="c6"><span class="c2">Gel-purify psb1A3 pcr, send off to sequencing w/ RFP F,R</span></li></ol><p class="c4"><span class="c0 c3">Friday</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Start liquid culture of psB1A7 </span></li></ol><ol class="c1"><li class="c6" value="1"><span class="c2">&nbsp;</span></li><li class="c6"><span class="c2">Digest - Debika (12-2 pm)</span></li><li class="c6"><span class="c2">Ligate, transform - Mitesh, Scott (5pm)</span></li></ol><p class="c4"><span class="c0 c3">Saturday</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Colony PCR of constructs (10 each)</span></li><li class="c6"><span class="c2">Mini-prep of psb1A7- Scott (9am)</span></li><li class="c6"><span class="c2">Run gel</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">hybB F, AOX R</span></li><li class="c8"><span class="c2">hybB F, ompA R</span></li><li class="c8"><span class="c2">ompA F, AOX R</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Need controls for everything from now on!</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Controls: (Can&rsquo;t read Scott&rsquo;s handwriting, sorry Scott! This stuff is still on the whiteboard in the lab)</span></p><p class="c4"><span class="c2">Ligate </span></p><ol class="c1"><li class="c6" value="1"><span class="c2">leave out a piece of the ligation so we know it won&rsquo;t work (- control)</span></li><li class="c6"><span class="c2">continue to use this throughout the transformation</span></li></ol><p class="c4"><span class="c2">Transformation control</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Just digest psB1A7 use it to transform (negative control</span></li></ol><hr><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">10/7/2010 (</span><span class="c0 c3">Thursday) </span></p><p class="c4"><span class="c2">GOALS:</span></p><p class="c4"><span class="c2">&nbsp;</span></p><ol class="c1"><li class="c6" value="2"><span class="c2">perform a heat shock transformation of psb1a7 -- this could not be done on Wed. because no plates were left to perform this. Had to make plates first. (completed Christina, Rob)</span></li></ol><ol class="c1"><li class="c6" value="3"><span class="c2">Mini-prep of psB1A3 (completed, Christina, Rob)</span></li><li class="c6"><span class="c2">send sequence w/ RFP F,R </span></li><li class="c6"><span class="c2">Gel-purify psb1A3 pcr, send off to sequencing w/ RFP F,R </span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">Make gel in Hammer Lab Chamber (the orange one) (gel completed, christina)</span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">Gel tray is max 80 or so ml </span></li><li class="c10"><span class="c2">perform gel extraction (I can do this in afternoon, Scott)</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Results and Observations from plates made on 10-6-10: </span></p><ol class="c1"><li class="c6" value="1"><span class="c2">psb1a3 had growth; no pink colonies (may be slight, can ask Ryan as she is expert)</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">made a starter culture from these plates and see if they turn pink. </span></li></ol><ol class="c1"><li class="c6" value="2"><span class="c2">psb1a7 had no growth - plates thrown out. </span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Protocols:</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Moved the plates of the psb1a7, psb1a3 transformations to fridge -- psb1A7 plates thrown out. (Scott)</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Make starter cultures from psb1A3/NB plates made on 10/6/10.(Christina)</span></p><p class="c4"><span class="c0 c3">&nbsp;</span></p><p class="c4"><span class="c0">perform a heat shock transformation of psb1a7 -- this could not be done on Wed. because no plates were left to perform this. Had to make plates first (Christina)</span></p><p class="c4 c13"><span class="c2">Add 1uL of resuspended</span><span class="c2 c3"> psb1A7</span><span class="c2"> in 10uL of competent cells (Novablue). (thawed on ice)</span></p><p class="c4 c13"><span class="c2 c11 c3">See Protocols page for Heat Shock Transformation </span></p><p class="c4 c13"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Mini-prep of psB1A3 from starter cultures grown on 9-6-10 (Christina)</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Autoclaved a variety of pipette tip boxes to stock up(Christina)</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">make a new 1% gel in the orange pcr chamber (the chamber is the casting tray -- GENIUS!) (Christina)</span></p><p class="c4"><span class="c2 c11 c3">See Protocols page for Preparing 1% Agarose Gels</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">PCR purifcation of psb1a3 pcr wiith RFP-F, RFP-R</span></p><p class="c4"><span class="c2 c11 c3">See Protocols page for PCR Purifacation</span></p><p class="c4"><span class="c0">&nbsp;</span></p><p class="c4"><span class="c0">Gel extraction of PCR from psb1a3 using primers RFP-F, RFP-R (Scott, Gita)</span></p><p class="c4"><span class="c2 c11 c3">See Protocols page for Gel Extraction</span></p><p class="c4"><span class="c2">Note: To elute DNA, added 30 &nbsp;&mu;L water (pH 7.0 &ndash; 8.5), let the column stand for 1 min, and then centrifuged for 1 min.</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">RE Digest Recipe for RFP-F3R (that Debika did this yesterday)(Christian is doing the digest today)</span></p><p class="c4"><span class="c2">12.5 uL H20 </span></p><p class="c4"><span class="c2">5uL 10X Promega Buffer B</span></p><p class="c4"><span class="c2">6 uL RFP-F3R (167.4 ng/uL 10.6.2010)</span></p><p class="c4"><span class="c2">5 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c4"><span class="c2">0.75 &nbsp;uL SpeI</span></p><p class="c4"><span class="c2">0.75 &nbsp;uL XmaI</span></p><p class="c4"><span class="c2 c3">Total=50 ul total</span></p><p class="c4"><span class="c2">Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)</span></p><p class="c4"><span class="c2">Start 5pm</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">10/8/2010</span></p><p class="c4"><span class="c0">Goals</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Nanospec the gel extract done on 10/7/2010 (of psb1a3 RFP-FR, 700 bp band)</span></li><li class="c6"><span class="c2">Run 4 uL of the gel extract on the gel and take picture (to prove psb1a3 has rfp!)</span></li><li class="c6"><span class="c2">Send off for sequencing:</span></li></ol><ol class=""><li class="c8" value="1"><span class="c2">gel extract with RFP FR</span></li><li class="c8"><span class="c2">the mini prep of psb1a3 with RFP FR (mini prep from thursday)</span></li></ol><ol class="c1"><li class="c6" value="4"><span class="c2">Repeat transformation of psb1a7!</span></li><li class="c6"><span class="c2">PCR purification of digest from (10/7/2010) of RFP F3R</span></li></ol><p class="c4 c13"><span class="c2 c11 c3">See Protocols page for PCR Purification</span></p><ol class="c5"><li class="c8" value="1"><span class="c2">run on gel to check for 700 bp band</span></li><li class="c8"><span class="c2">if ok, then </span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">ligate to Hyb.ompa and psb1a3 or psb1a7 (which vector)?</span></li></ol><p class="c4"><span class="c0">Observations</span></p><p class="c4"><span class="c2">The transformation of psb1a7 into NB did not work. We should check whether the strain is compatible with the plasmid. &nbsp;</span></p><p class="c4"><span class="c2">The colonies from the transformation of psb1a3 directly from well into NB have turned slightly pink. </span></p><p class="c4"><span class="c2">Liquid cultures of psb1a3 colonies from plates from 10.6.2010 appear brown with no pink. However, when we pelleted them for miniprep, the pellet was pink!</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">minprep of psb1a3 liq culture from palte from10.6.2010</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Transformation of pSB1A7 in NB cells (with + control)</span></p><p class="c4"><span class="c2">Megan suggested we use fresh NB cells (from Gaucher cells), use a positive control (known good plasmids)</span></p><p class="c4"><span class="c2 c11">2 reactions:</span></p><p class="c4"><span class="c2">10 &#1405;L Nova Blue cells + 5 &#1405;L of mRFP from Gaucher Lab (labeled mRFP mini prep 3/11/09)</span></p><p class="c4"><span class="c2">10 &#1405;L Nova Blue cells + 2&#1405;L of psb1a7 from &nbsp;well aliquot</span></p><p class="c4"><span class="c2"> </span></p><p class="c4"><span class="c2 c11 c3">See Protocols page for Heat Shock Transformation.</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">PCR purification of digest from (10/7/2010) of RFP F3R (Gita)</span></p><p class="c4"><span class="c2 c11 c3">See Protocols page for PCR Purification.</span></p><p class="c4"><span class="c2">notes: elute in 30 uL!</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Nanospec</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">purified digest of RFP-F3R (labeled with 10.8.2010) = 18.1 ng/uL</span></li><li class="c6"><span class="c2">Gel extract of RFP FR (tube labeled 1, date 10.7.2010) = 84 ng/uL</span></li><li class="c6"><span class="c2">Gel extract of RFP FR (tube labeled 2, date 10.7.2010) = 91.3 ng/uL</span></li><li class="c6"><span class="c2">psb1a3 mini prep </span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">tube 1: 64 ng/uL</span></li><li class="c8"><span class="c2">tube 2 :56 ng/uL</span></li></ol><p class="c4"><span class="c0">&nbsp;</span></p><p class="c4"><span class="c0">Run on 1% gel</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Gel extractions from 10.7.2010 of psb1a3+ RFP-FR (2 tubes)</span></li><li class="c6"><span class="c2">purified, digested RFP-F3R from today, 10.8.2010</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">For tomorrow</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">high priority</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">pick colonies of psb1a7 (if succesful) and transfer to liquid culture</span></li><li class="c8"><span class="c2">tranform cells using psb1c3, since it is the vector we have to submit in. </span></li><li class="c8"><span class="c2">ligate RFP F3R to hyb.ompa and psb1a7 (likely have to be done sunday)</span></li></ol><ol class="c1"><li class="c6" value="2"><span class="c2">autoclave more water</span></li><li class="c6"><span class="c2">Find some chloramphenicol since we have to submit parts in psb1c3 and it has chloramphenicol resistance</span></li><li class="c6"><span class="c2">low priority</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">run pcr on psb1a3 minipreps today to check for rfp</span></li></ol><p class="c4 c13"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">10/9/2010</span></p><p class="c4"><span class="c0">Goals</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Troubleshoot psb1a7, psb1a3</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">List of wells, inserts, and sequences (</span><span class="c0"><a href="http://www.google.com/url?q=http%3A%2F%2Fpartsregistry.org%2Fassembly%2Flibraries.cgi%3Fid%3D31&amp;sa=D&amp;sntz=1&amp;usg=AFQjCNG0fXu0QjGrIZuxeCNGIxGvHiHArw">http://partsregistry.org/assembly/libraries.cgi?id=31</a></span><span class="c2">)</span></li></ol><p class="c4"><span class="c0">Notes</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Troubleshoot psb1a7</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">does it have a cbbd toxic gene in it?</span></li></ol><ol class=""><li class="c10" value="1"><span class="c2">No, it has an insert coding for a antibody chain, part: K126000</span></li></ol><p class="c4 c15"><span class="c2">(</span><span class="c0"><a href="http://www.google.com/url?q=http%3A%2F%2Fpartsregistry.org%2Fwiki%2Findex.php%3Ftitle%3DPart%3ABBa_K126000&amp;sa=D&amp;sntz=1&amp;usg=AFQjCNFBJXz_hFsVsi4FZU0lrYCcPps-HA">http://partsregistry.org/wiki/index.php?title=Part:BBa_K126000</a></span><span class="c2">) </span><span class="c0"><a href="">(http://partsregistry.org/assembly/plates.cgi?id=1259)</a></span></p><p class="c4"><span class="c2">&nbsp;</span></p><ol class="c5"><li class="c8" value="2"><span class="c2">can we digest (is it digested) and ligate it directly from the well to our parts?</span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">should we pcr it first?</span></li></ol><ol class="c5"><li class="c8" value="3"><span class="c2">do we have a ccdb tolerant strain?</span></li></ol><p class="c4 c13"><span class="c2">&nbsp;</span></p><ol class="c1"><li class="c6" value="2"><span class="c2">Trobuleshoot psb1a3</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">psb1a3 has RFP </span><span class="c0"><a href="">(http://partsregistry.org/partsdb/get_part.cgi?part=pSB1A3)</a></span><span class="c2"> </span></li><li class="c8"><span class="c2">BBa_J04450 = RFP</span></li></ol><ol class="c1"><li class="c6" value="3"><span class="c2">tranform cells using psb1c3, since it is the vector we have to submit in. </span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">Have plasmid backbone from kit (Where is it?)</span></li><li class="c8"><span class="c2">Has RFP insert for in well Plate 1 - A3 (</span><span class="c0"><a href="http://www.google.com/url?q=http%3A%2F%2Fpartsregistry.org%2Fassembly%2Fplates.cgi%3Fid%3D1257&amp;sa=D&amp;sntz=1&amp;usg=AFQjCNGZEL21q96T5macGA-sc1m3waRKlQ">http://partsregistry.org/assembly/plates.cgi?id=1257</a></span><span class="c2">)</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Protocols</span></p><p class="c4"><span class="c2"></span><span class="c0">RE Double Digest Recipe for pSB1A3 (from 10/8/2010)</span></p><p class="c4"><span class="c2">6.5 uL H20 </span></p><p class="c4"><span class="c2">3uL 10X Promega Buffer E </span></p><p class="c4"><span class="c2">16 uL pSB1A3 (10.8.2010, 64 ng/uL)(tube 1)</span></p><p class="c4"><span class="c2">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c4"><span class="c2">0.75 uL SpeI</span></p><p class="c4"><span class="c2">0.75 uL EcoRI</span></p><p class="c4"><span class="c2 c3">Total=30 ul total</span></p><p class="c4"><span class="c2">Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)</span></p><p class="c4"><span class="c2">Predict a insert (1000 bp) and plasmid (2000 bp)</span></p><p class="c4"><span class="c0">RE Digest Recipe for pSB1A3 (from 10/8/2010)</span></p><p class="c4"><span class="c2">5.0 uL H20 </span></p><p class="c4"><span class="c2">2.5 uL 10X Promega Buffer E </span></p><p class="c4"><span class="c2">16 uL pSB1A3 (10.8.2010, 50 ng/uL)(tube 2)</span></p><p class="c4"><span class="c2">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c4"><span class="c2">0.75 uL SpeI</span></p><p class="c4"><span class="c2 c3">Total=30 ul total</span></p><p class="c4"><span class="c2">Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)</span></p><p class="c4"><span class="c2">Digest start::12pm</span></p><p class="c4"><span class="c0">For Tomorrow:</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">PREFORM PCR on WHITE colonies to check for what is in them: triple ligation plates from Oct </span></li><li class="c6"><span class="c2">Ask Gaucher lab if they have 1) ccdb resistance strains 2) vector lacking promoters and have amp resistance 3) the three tubes of plasmid backbone from the standard iGem kit</span></li><li class="c6"><span class="c2">Need more Promega Buffer E</span></li></ol><p class="c4"><span class="c2 c11">&nbsp;</span></p>
+
<title>10/3-10/9</title><style type="text/css">ol{margin:0;padding:0}p{margin:0}.c0{color:#ffffff;font-size:10pt;text-decoration:underline;font-family:Arial}.c8{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:72.0pt}.c6{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:36.0pt}.c10{padding-left:0pt;line-height:1.15;direction:ltr;margin-left:108.0pt}.c2{color:#ffffff;font-size:10pt;font-family:Arial}.c9{line-height:1.15;text-indent:36.0pt;direction:ltr}.c4{line-height:1.15;text-indent:0pt;direction:ltr}.c15{margin-left:72.0pt}.c7{text-align:center}.c16{list-style-type:lower-latin}.c1{list-style-type:disc}.c19{background-color:#ffffff}.c17{list-style-type:lower-roman}.c12{list-style-type:square}.c11{font-style:italic}.c14{list-style-type:decimal}.c3{font-weight:bold}.c18{margin-left:108.0pt}.c13{margin-left:36.0pt}.c20{border-collapse:collapse}.c5{list-style-type:circle}</style></head><body class="c19"><p class="c4"><span class="c2 c3">10/3/10</span></p><p class="c4"><span class="c0">For Today:</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Run on a gel</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">aoxa, aoxb next to the troubleshooting pcrs from today, specifically of the PCRS using Ompa-F and Aoxa/b-R since they are similar sizes</span></li></ol><ol class="c1"><li class="c6" value="2"><span class="c2">Take out plates from incubator. Record observations</span></li></ol><ol class=""><li class="c8" value="1"><span class="c2">Run colony PCRs on at least 20 colonies per construct (yes 20)</span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">Check which primers to use for colony pcr! We can likely use the primers we already have</span></li><li class="c10"><span class="c2">Run results on gel</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Results: plated colonies from 10/2/10 (Mitesh &amp; Rob):</span></p><p class="c4"><span class="c2">Observed that HybB-ompA-Aox1(a/b) constructs were successfully transformed- strong colony growth and red colonies (strange- given that these were supposedly constructs without RFP). The color strongly suggests RFP. Red colony patterns show no consistent trends- some are densely red in the middle of the plate, others mainly around the edges- red intensity varied widely even on the same plate. </span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4 c7"><img height="268.0" src="http://2010.igem.org/wiki/images/2/28/10-3a.png" width="203.0"></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Colony PCR of HybB-ompA-Aox constructs</span></p><p class="c4"><span class="c2">Picked colonies from plates of HybB-ompA-Aox-RFP and added to 25 ul of water each,10 colonies from Aox1a containing plates, and 10 from Aox1b containing plates:</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">HybB-ompA-Aox1a-RFP: 1.5 mL Tubes 1-10</span></p><p class="c4"><span class="c2">HybB-ompA-Aox1b-RFP: 1.5 mL Tubes 11-20 </span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Used 5 ul for PCR, and added 250 ul of LB media to the rest. </span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Colony PCRs (for each colony): (Rob, Mitesh &amp; Scott)</span></p><p class="c4"><span class="c2">5 uL of cells</span></p><p class="c4"><span class="c2">25.5 uL H2O</span></p><p class="c4"><span class="c2">10 uL GOTAQ</span><span class="c2 c3"> 5X Reaction buffer</span></p><p class="c4"><span class="c2">5 uL HybB-F forward primer</span></p><p class="c4"><span class="c2">5 uL RFP-R reverse primer</span></p><p class="c4"><span class="c2">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c4"><span class="c2">0.5 uL polymerase enzyme, TAQ</span></p><p class="c4"><span class="c2 c3">Total Volume= 50 uL</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Colony PCRs (for each colony):</span></p><p class="c4"><span class="c2">5 uL of cells</span></p><p class="c4"><span class="c2">25.5 uL H2O</span></p><p class="c4"><span class="c2">10 uL GOTAQ</span><span class="c2 c3"> 5X Reaction buffer</span></p><p class="c4"><span class="c2">5 uL HybB-F forward primer</span></p><p class="c4"><span class="c2">5 uL Aox1a or b-R reverse primer</span></p><p class="c4"><span class="c2">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c4"><span class="c2">0.5 uL polymerase enzyme, TAQ</span></p><p class="c4"><span class="c2 c3">Total Volume= 50 uL</span></p><p class="c4"><span class="c2 c3"> </span></p><p class="c4"><span class="c2">Important: For the PCR tubes, the ones labeled 1,3,5,7,9 are treated with Aox1a reverse primer.</span></p><p class="c4"><span class="c2">All the even number (2,4,6,...16,18,20) labeled have RFP-R reverse primer in them. And the tubes labeled 11,13,15,17,19 have Aox 1bR reverse primer in them.</span></p><p class="c4"><span class="c2">PCR start time: 6 PM</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">For Monday:</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Check colony pcr on a gel</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">calculate the predicted sizes and compare to gel results</span></li></ol><ol class="c1"><li class="c6" value="2"><span class="c2">Continue with colony PCRS if more info is required</span></li><li class="c6"><span class="c2">Check triple ligations on gel</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">10/4/2010</span></p><p class="c4"><span class="c0">Protocols</span></p><p class="c4"><span class="c0">&nbsp;</span></p><p class="c4"><span class="c0">1 % Gel to Check colony PCRs from 10/3/2010</span></p><p class="c4"><span class="c2">Started at 9 am</span></p><p class="c4"><span class="c2">Order:ladder:1-6|ladder|7-12|ladder|13-14</span></p><p class="c4"><span class="c2">Important: For the PCR tubes, the ones labeled 1,3,5,7,9 are treated with Aox1a reverse primer.</span></p><p class="c4"><span class="c2">All the even number (2,4,6,...16,18,20) labeled have RFP-R reverse primer in them. And the tubes labeled 11,13,15,17,19 have Aox 1bR reverse primer in them.</span></p><p class="c4"><span class="c2">PCR start time: 6 PM</span></p><p class="c4"><span class="c2">gel start at 10:34 am</span></p><p class="c4"><img height="329.0" src="http://2010.igem.org/wiki/images/a/a0/10-3c.png" width="440.0"></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Predicted Bands:</span></p><p class="c4"><span class="c2">Hyb-F + RFP-R approx 2100-2200 &nbsp;bp</span></p><p class="c4"><span class="c2">Hyb-F + Aoxa-R - approx 1500</span></p><p class="c4"><span class="c2">Hyb-F + Aoxb-R approx 1500</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Observations- 2200 bp band is seen, but other bands are also observed for the Hyb-F and RFP-R reactions</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Suggestions:</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Troubleshoot by PCR sequencing the basic products (e.g. sequence just HybB in one tube, in the next, just OmpA; in the next, just AOX; in the next, just RFP). This will tell us whether each gene is actually present in the cells PCRed.</span></li><li class="c6"><span class="c2">Start over again entirely, from the very beginning, digesting each individual gene, ligating, transforming.</span></li><li class="c6"><span class="c2">Start using controls. When possible, include positive and negative controls. </span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">For example, when doing a double-ligation, for instance, perform experiment alongside designed to fail (e.g. lacking one of the ligation genes) for a negative control. </span></li><li class="c8"><span class="c2">To see if the plasmid pSB1A3 is causing our problems, transformed this plasmid alone directly into E. coli and plate.</span></li></ol><ol class="c1"><li class="c6" value="4"><span class="c2">If pSB1A3 turns out to be the problem, can attempt to use a different plasmid backbone. We&rsquo;ve researched and found plasmid pSB1C3 to have the desired characteristics, including lack of a &nbsp;promoter, beginning EcoRI and ending SpeI restriction sites.</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Plan to go forward:</span></p><p class="c4"><span class="c2">1. Transform &ldquo;plain&rdquo; plasmid pSB1A3 into E. coli today 4 OCT 2010. Check for RFP expression.</span></p><p class="c4"><span class="c2">2. In tandem with step 3, troubleshoot by sequencing the basic gene products in the &ldquo;bad&rdquo; HybB-OmpA-AOX contsructs, looking specifically for each gene.</span></p><p class="c4"><span class="c2">3. In tandem with step 2, re-do the entire construction process from the individual genes up.</span></p><p class="c4"><span class="c2">4. </span><span class="c2 c3">Transform cells using new biobrick vectors, like psb1c3, and use those instead of psb1a3!</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">NOTE: Need to make more media plates!</span></p><p class="c4"><span class="c2 c3">&nbsp;</span></p><p class="c4"><span class="c0">Protocols</span></p><p class="c4"><span class="c0">&nbsp;</span></p><p class="c4"><span class="c0">Transformation of NB cells using pSB1a3</span></p><p class="c4"><span class="c2">10 &#1405;L Nova Blue cells + 5 &#1405;L of pSB1A3 from MiniPrep</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">1. Left cells and plasmid on ice.</span></p><p class="c4"><span class="c2">2. Added plasmid (5&#1405;L) to cells. Mix gently by swirling pipette tip in mixture (DO NOT ASPIRATE).</span></p><p class="c4"><span class="c2">3. Left cells on ice for 30 min.</span></p><p class="c4"><span class="c2">4. Applied heat shock of 45 seconds in 42C bath.</span></p><p class="c4"><span class="c2">5. Put tubes on ice for 2 min.</span></p><p class="c4"><span class="c2">6. Added 250 &#1405;L of LB (room temp.)</span></p><p class="c4"><span class="c2">7. Incubated 1 hour at 37 C</span></p><p class="c4"><span class="c2">8. Plated 100&#1405;L (x2 plates) and left plates in the 37 degrees incubator &nbsp;</span></p><p class="c4"><span class="c2">9. Incubated overnight at 37C. </span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Troubleshooting PCRs</span></p><p class="c4"><span class="c0">For each test (e.g. HybB, OmpA, AOX1a, etc.)</span></p><p class="c4"><span class="c2">2 uL of cells</span></p><p class="c4"><span class="c2">9.4 uL H2O</span></p><p class="c4"><span class="c2">4 uL GOTAQ</span><span class="c2 c3"> 5X Reaction buffer</span></p><p class="c4"><span class="c2">2 uL forward primer</span></p><p class="c4"><span class="c2">2 uL reverse primer</span></p><p class="c4"><span class="c2">0.4 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></p><p class="c4"><span class="c2">0.2 uL polymerase enzyme, GOTAQ</span></p><p class="c4"><span class="c2 c3">Total Volume= 20 uL &nbsp;</span></p><p class="c4"><span class="c2 c3">&nbsp;</span></p><p class="c4"><span class="c0">Combinations</span></p><p class="c4"><span class="c2 c3">A</span><span class="c2">: HybB-OmpA-AOX1a-pSB1A3 in NovaBlue cells, ligated 2 OCT 2010 (Margo + Scott)</span></p><p class="c9"><span class="c2">Selected 1 </span><span class="c2 c3">red</span><span class="c2"> colony, suspended in 20 uL sterile dH2O</span></p><p class="c9"><span class="c2">1: HybB forward &amp; reverse primers</span></p><p class="c9"><span class="c2">2: OmpA forward &amp; reverse primers</span></p><p class="c9"><span class="c2">3: AOX1a forward &amp; reverse primers</span></p><p class="c9"><span class="c2">4: RFP forward &amp; reverse primers</span></p><p class="c9"><span class="c2">&nbsp;</span></p><p class="c9"><span class="c2">Selected 1 </span><span class="c2 c3">white</span><span class="c2"> colony, suspended in 20 uL sterile dH2O</span></p><p class="c9"><span class="c2">5: HybB forward &amp; reverse primers</span></p><p class="c9"><span class="c2">6: OmpA forward &amp; reverse primers</span></p><p class="c9"><span class="c2">7: AOX1a forward &amp; reverse primers</span></p><p class="c9"><span class="c2">8: RFP forward &amp; reverse primers</span></p><p class="c9"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">B</span><span class="c2">: HybB-OmpA-AOX1b-pSB1A3 in NovaBlue cells, ligated 2 OCT 2010 (Margo + Scott)</span></p><p class="c9"><span class="c2">Selected 1 </span><span class="c2 c3">red</span><span class="c2"> colony, suspended in 20 uL sterile dH2O</span></p><p class="c9"><span class="c2">1: HybB forward &amp; reverse primers</span></p><p class="c9"><span class="c2">2: OmpA forward &amp; reverse primers</span></p><p class="c9"><span class="c2">3: AOX1a forward &amp; reverse primers</span></p><p class="c9"><span class="c2">4: RFP forward &amp; reverse primers</span></p><p class="c9"><span class="c2">&nbsp;</span></p><p class="c9"><span class="c2">Selected 1 </span><span class="c2 c3">white</span><span class="c2"> colony, suspended in 20 uL sterile dH2O</span></p><p class="c9"><span class="c2">5: HybB forward &amp; reverse primers</span></p><p class="c9"><span class="c2">6: OmpA forward &amp; reverse primers</span></p><p class="c9"><span class="c2">7: AOX1a forward &amp; reverse primers</span></p><p class="c9"><span class="c2">8: RFP forward &amp; reverse primers</span></p><p class="c4"><span class="c2 c3">&nbsp;</span></p><p class="c4"><span class="c2 c3">P</span><span class="c2">: &ldquo;plain&rdquo; mini-prepped pSB1A3 plasmid</span></p><p class="c4"><span class="c2">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;RFP forward &amp; reverse primers</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Ran a PCR on the samples using 1KB ladder -- ran A 1-7</span></p><p class="c4"><span class="c2">Lane &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1: ladder</span></p><p class="c9"><span class="c2">2: A1</span></p><p class="c9"><span class="c2">3: A2</span></p><p class="c9"><span class="c2">4: A3</span></p><p class="c9"><span class="c2">5: A4</span></p><p class="c9"><span class="c2">6: A5</span></p><p class="c9"><span class="c2">7: A6</span></p><p class="c9"><span class="c2">8: A7</span></p><p class="c9"><span class="c2">&nbsp;</span></p><p class="c4"><img height="297.0" src="http://2010.igem.org/wiki/images/5/57/10-3b.png" width="392.0"></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">Note: need to make a big batch of 1% gels tomorrow since we are running so many samples.</span></p><p class="c4"><span class="c2 c3">Need more agarose from Gaucher Lab. COMPLETED</span></p><p class="c4"><span class="c2 c3">Need more EtBr. COMPLETED</span></p><p class="c4"><span class="c2 c3">Need a 100bp Ladder to see some of this stuff (small constructs) -- we might need to get Ryan to order some. </span></p><p class="c4"><span class="c2 c3">&nbsp;</span></p><p class="c4"><span class="c2 c3">10-5-10</span></p><p class="c4"><span class="c2">Obtain some more agarose and EtBr from Gaucher lab.</span></p><p class="c4"><span class="c2">Make 1% gels. </span></p><p class="c4"><span class="c2 c11 c3">See Protocols page for Preparing 1% Agarose Gels</span></p><p class="c4"><span class="c2">Ran a gel on the remaining PCR samples from 10-4-10</span></p><p class="c4"><span class="c2">Following 1 KB ladder, lanes should read in order:</span></p><p class="c4"><span class="c2">A8, P, B1, B2, B3, B4, B5, B6, B7, B8. </span></p><p class="c4"><span class="c2">No digital picture available, but one was printed and is in the lab, labelled with the date. </span></p><p class="c4"><span class="c2">&nbsp;</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Starting construct building strategy over again</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">Reactions:</span></li></ol><ol class="c14"><li class="c6" value="1"><span class="c2">HyBb F,R (template biobrick)</span></li><li class="c6"><span class="c2">OmpA F,R (template ompa vector)</span></li><li class="c6"><span class="c2">Aox1a F,R (template synthesized gene)</span></li><li class="c6"><span class="c2">Aox1a F,R2 (template synthesized gene)</span></li><li class="c6"><span class="c2">Aox1b F,R (template synthesized gene)</span></li><li class="c6"><span class="c2">Aox1b F,R2 (template synthesized gene)</span></li><li class="c6"><span class="c2">RFP F,R (template RFP plasmid) (from 9.27.2010 miniprep)</span></li><li class="c6"><span class="c2">RFP F2,R &nbsp;(template RFP plasmid) (from 9.27.2010 miniprep)</span></li></ol><p class="c4 c13"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">PCR Protocol (Scott)</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">26.5 uL H2O</span></li><li class="c6"><span class="c2">10 uL </span><span class="c2 c3">PHUSION 5X Reaction buffer</span></li><li class="c6"><span class="c2">5 uL forward primer</span></li><li class="c6"><span class="c2">5 uL reverse primer</span></li><li class="c6"><span class="c2">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></li><li class="c6"><span class="c2 c3">2 uL</span><span class="c2"> template DNA</span></li><li class="c6"><span class="c2">0.5 uL polymerase enzyme, </span><span class="c2 c3">PHUSION</span></li></ol><p class="c4"><span class="c2 c3">Total Volume= 50 uL</span></p><p class="c4"><span class="c2">started 4:33pm</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">10/6/2010</span></p><p class="c4"><span class="c0">Goals</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">The gel from 10/5/2010 of the troubleshooting pcrs, specifically the reaction of pSB1A3 with RFP-F and R, suggests that the vector used for ligation has RFP. We will use anothe rvector from biobrick</span></li><li class="c6"><span class="c2">We want to transform cells with the new biobrick vector today, miniprep the cells tomorrow morning, digest, and ligate to our hyb.ompa+aox that we have or are making now. Finish by friday is the goal. </span></li><li class="c6"><span class="c2">Sequence hyb-RFP (for bronze medal requirements)</span></li><li class="c6"><span class="c2">begin hyb-ompa-rfp building when primers come in</span></li><li class="c6"><span class="c2">I saw Richard&rsquo;s sequence was for psb1c3, but we are using psb1a3; this may a primer and digests! Investigate!</span></li><li class="c6"><span class="c2">Sequence white colonies from the plates on 10/3/2010</span></li><li class="c6"><span class="c2">For today:</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">Reconstitute new biobrick vector (psb1A7) COMPLETED</span></li><li class="c8"><span class="c2">Transform NB cells using new biobrick vector COMPLETED</span></li><li class="c8"><span class="c2">Check PCRs from 10/5/2010 on gel (tubes in yellow pcr box labeled 1-8 and dated 10.5) COMPLETED</span></li><li class="c8"><span class="c2">Make Amp plates</span></li><li class="c8"><span class="c2">Autoclave pipette tips</span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">This involves testing which tips are compatible with ours (we have lots of random tips)</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Notes on Vector-</span></p><p class="c4"><span class="c2">Here is an experience from another team concerning transcription of a product during an &ldquo;off state&rdquo;:</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Our team (</span><span class="c0"><a href="http://www.google.com/url?q=http%3A%2F%2Fpartsregistry.org%2Fwiki%2Findex.php%2FDavidson_2006&amp;sa=D&amp;sntz=1&amp;usg=AFQjCNG5h0TpmlIOGm0emKQ96Nz1nWxszw">Davidson College</a></span><span class="c2">) has used this vector in </span><span class="c2 c11">E. coli</span><span class="c2"> strain JM109 extensively. Clones are Amp resistant and are recovered in reasonable yields from mini preps. One drawback is that the vector has read-through transcription that comes into the cloned BioBrick part from both directions. This problem is a major one when an &quot;off state&quot; is critical for the control of a device. Parts without a promoter are expressed in this vector, regardless of orientation (see</span><span class="c2"><a href="http://www.google.com/url?q=http%3A%2F%2Fpartsregistry.org%2Fwiki%2Findex.php%2FPart%3ABBa_J31009%3ADesign&amp;sa=D&amp;sntz=1&amp;usg=AFQjCNEbgpV7QGpUM3sHpHi6sMXb_tKAoA"> </a></span><span class="c0"><a href="http://www.google.com/url?q=http%3A%2F%2Fpartsregistry.org%2Fwiki%2Findex.php%2FPart%3ABBa_J31009%3ADesign&amp;sa=D&amp;sntz=1&amp;usg=AFQjCNEbgpV7QGpUM3sHpHi6sMXb_tKAoA">pSB1A7 Part Design</a></span><span class="c2"> for details). Reverse read-through might be from the Amp resistance gene. Forward read-through might be from a cryptic promoter, since no known genes in the backbone are in the plus orientation.</span></p><p class="c4"><span class="c2">--</span><span class="c0"><a href="http://www.google.com/url?q=http%3A%2F%2Fpartsregistry.org%2FUser%3AKahaynes&amp;sa=D&amp;sntz=1&amp;usg=AFQjCNGORq4D7Hq4fMg0TPGjwS-hTXsGSQ">Kahaynes</a></span><span class="c2"> 15:48, 23 October 2006 (EDT)</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Alternative vectors:</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">pSB1A7 (2010 Kit plate 3, 15G well, name BBa_K126000)</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">Reasons:</span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">There are double terminators on either side of the multiple cloning site that block any read through or unwanted transcription from the plasmid promoters (plasmid promoters are involved in antibiotic production) </span></li><li class="c10"><span class="c2">Ampicillin resistance</span></li><li class="c10"><span class="c2">Already in kit</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><table cellpadding="0" cellspacing="0" class="c20"><tbody></tbody></table><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Protocols</span></p><p class="c4"><span class="c2">1. Resuspend </span><span class="c2 c3">psb1A7</span><span class="c2"> DNA in 10uL of autoclaved water. </span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4 c13"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">3. Run gels of PCR&rsquo;s on 10-5-10. </span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><img height="330.0" src="http://2010.igem.org/wiki/images/2/29/10-3d.png" width="441.0"></p><p class="c4"><span class="c2">Order:</span></p><ol class="c14"><li class="c6" value="1"><span class="c2">HyBb F,R (template biobrick)</span></li><li class="c6"><span class="c2">OmpA F,R (template ompa vector)</span></li><li class="c6"><span class="c2">Aox1a F,R (template synthesized gene)</span></li><li class="c6"><span class="c2">Aox1a F,R2 (template synthesized gene)</span></li><li class="c6"><span class="c2">Aox1b F,R (template synthesized gene)</span></li><li class="c6"><span class="c2">Aox1b F,R2 (template synthesized gene)</span></li><li class="c6"><span class="c2">RFP F,R (template RFP plasmid) (from 9.27.2010 miniprep)</span></li><li class="c6"><span class="c2">RFP F2,R &nbsp;(template RFP plasmid) (from 9.27.2010 miniprep)</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Growing colony of pSB1a3 transformed cells (Scott)</span></p><p class="c4"><span class="c2">I picked 3 colonies from the plate marked &ldquo;MFC 9.15.2010 C1 &ldquo; from 9.15.2010, with the psb1a3 transformed cells that are slighty pink. I did 3 mL + 3 uL of Carb, and put them in the incubator. </span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">PCR of pSB1A3 (direct from well aliquot) using RFP-F,R</span></p><ol class="c1"><li class="c6" value="8"><span class="c2">27.5 uL H2O</span></li><li class="c6"><span class="c2">10 uL </span><span class="c2 c3">GoTaq 5X Reaction buffer</span></li><li class="c6"><span class="c2">5 uL forward primer (RFP-F) </span></li><li class="c6"><span class="c2">5 uL reverse primer (RFP-R)</span></li><li class="c6"><span class="c2">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></li><li class="c6"><span class="c2 c3">1 uL</span><span class="c2"> template DNA</span></li><li class="c6"><span class="c2">0.5 uL polymerase enzyme, </span><span class="c2 c3">GoTaq</span></li></ol><p class="c4"><span class="c2 c3">Total Volume= 50 uL</span></p><p class="c4"><span class="c2"> Left in block B- Thursday people need to retieve it- Need to gel purify- scroll down to see a lit of things to do for Thursday. These steps are also highligted in blue below. </span></p><p class="c4"><span class="c0">PCR of mRFP miniprep (direct from well aliquot) using RFP-F3,R</span></p><ol class="c1"><li class="c6" value="15"><span class="c2">26.5 uL H2O</span></li><li class="c6"><span class="c2">10 uL </span><span class="c2 c3">GoTaq 5X Reaction buffer</span></li><li class="c6"><span class="c2">5 uL forward primer (RFP-F3) </span></li><li class="c6"><span class="c2">5 uL reverse primer (RFP-R)</span></li><li class="c6"><span class="c2">1 uL dNTP 10 mM - (thawed &amp; kept on ice)</span></li><li class="c6"><span class="c2 c3">2 uL</span><span class="c2"> template DNA</span></li><li class="c6"><span class="c2">0.5 uL polymerase enzyme, </span><span class="c2 c3">GoTaq</span></li></ol><p class="c4"><span class="c2 c3">Total Volume= 50 uL</span></p><p class="c4"><span class="c2">Left in block B- Thursday people need to retieve it &nbsp;- Scott knows what to do with this. I don&rsquo;t. -Debika</span></p><p class="c4"><span class="c0">&nbsp;</span></p><p class="c4"><span class="c0">&nbsp;</span></p><p class="c4"><span class="c0">Transformation of NB using pSB1A3 direct from Gel aliquot) -</span><span class="c0 c3">completed (Christina, Debika)</span></p><p class="c4"><span class="c2">10 &#1405;L Nova Blue cells + 5 &#1405;L of pSB1A3 from MiniPrep</span></p><p class="c4"><span class="c2"> &nbsp;</span></p><p class="c4"><span class="c2">1. Left cells and plasmid on ice.(Scott)</span></p><p class="c4"><span class="c2">2. Added plasmid (5&#1405;L) to cells. Mix gently by swirling pipette tip in mixture (DO NOT ASPIRATE).</span></p><p class="c4"><span class="c2">3. Left cells on ice for 30 min.</span></p><p class="c4"><span class="c2">4. Applied heat shock of 45 seconds in 42C bath. (Debika)</span></p><p class="c4"><span class="c2">5. Put tubes on ice for 2 min. (I messed up and put in LB in a minute instead of 2 minutes. Left it in ice again for a minute after adding LB). Don&rsquo;t know how that will affect our results. Sorry if this transformation doesn&rsquo;t work). </span></p><p class="c4"><span class="c2">6. Added 250 &#1405;L of LB (room temp.)</span></p><p class="c4"><span class="c2">7. Incubated 1 hour at 37 C</span></p><p class="c4"><span class="c2">8. Plated 100&#1405;L (x2 plates) and left plates in the 37 degrees incubator. (Christina) &nbsp;</span></p><p class="c4"><span class="c2">9. Incubated overnight at 37C. &nbsp;</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Transformation of NB using pSB1A7 direct from Gel aliquot) -</span><span class="c0 c3">completed (Christina)</span></p><p class="c4"><span class="c2">10 &#1405;L Nova Blue cells + 5 &#1405;L of pSB1A3 from MiniPrep</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Add 1uL of resuspended</span><span class="c2 c3"> psb1A7</span><span class="c2"> in 10uL of competent cells (Novablue). (thawed on ice)</span></li></ol><p class="c4"><span class="c2 c11 c3">See Protocols page for Heat Shock Transformation </span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Note: The incubation step was skipped, which likely will lead to little growth</span></p><hr><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">Meeting Notes</span></p><p class="c4"><span class="c0 c3">Priority List</span></p><p class="c9"><span class="c2 c11">Critical</span></p><ol class="c16"><li class="c8" value="1"><span class="c2">hyb.ompa.aox construct (in pSB1A7)</span></li><li class="c8"><span class="c2">hyb.ompa.RFP construct (in pSB1A7) (Scott)</span></li><li class="c8"><span class="c2">Sequencing of constructs and conformation with standard</span></li><li class="c8"><span class="c2">Biobrick</span></li></ol><p class="c4 c13"><span class="c2 c11">Need </span></p><ol class="c16"><li class="c8" value="1"><span class="c2">Calorimetric/Heat &nbsp;(Rob, Christian)</span></li><li class="c8"><span class="c2">Periplasmic Expression Picture (Fluorescent Microscope) (Scott)</span></li><li class="c8"><span class="c2">Cold Shock Quantification (hybB)</span></li><li class="c8"><span class="c2">Modeling (Gita, Mitesh)</span></li></ol><ol class="c17"><li class="c10" value="1"><span class="c2">Liquid culture</span></li><li class="c10"><span class="c2">Solid Media</span></li></ol><p class="c4"><span class="c2">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span><span class="c2 c11">Want</span></p><ol class="c16"><li class="c8" value="1"><span class="c2">Confirm pSB1A3 has RFP in it</span></li></ol><ol class="c17"><li class="c10" value="1"><span class="c2">Pick colony from plates, liquid culture. (COMPLETED)(SCOTT) &nbsp;&ldquo;MFC 9.15.2010 C1 &ldquo;</span></li></ol><p class="c4 c18"><span class="c2">Thursday: Mini-prep, off to sequencing. Ask Ryan or Megan</span></p><p class="c4 c15"><span class="c2 c11"> &nbsp; &nbsp; ii &nbsp; &nbsp; </span><span class="c2">Aliquot from well, PCR (COMPLETED, DEBIKA, left in right side block overnight)</span></p><p class="c4 c18"><span class="c2">Thursday: gel purify, off to sequencing. Ask Ryan or Megan</span></p><hr><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">Detailed Plan</span></p><p class="c4"><span class="c0 c3">Today (wed)</span></p><p class="c4"><span class="c2 c11">Critical Actions</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">hyb.ompa.rfp construct</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">Start PCR of RFP using new primer using a plasmid with ryan&rsquo;s rfp (pcold and rfp, or the gaucher lab rfp plasmid) (Debika, completed) </span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c11">Want Actions</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">confirm PSB1A3 has RFP</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">Scott</span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">Pick colony from original psb1a3 plates &nbsp;&ldquo;MFC 9.15.2010 C1 &ldquo; and put in liq culture (completed)</span></li></ol><ol class=""><li class="c8" value="2"><span class="c2">Debika</span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">Start PCR of psb1a3 directly from well, using primers RFP-F and RFP-R (Debika, completed)</span></li></ol><ol class=""><li class="c8" value="3"><span class="c2">Christina, Debika,</span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">transformation of NB cells using original psb1a3 aliquot (completed)</span></li></ol><p class="c4 c15"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c11">Other Actions</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">order psb1a7 sequencing primers (the forward psb1a3-F primer is ok, but we need a reverse primer)</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0 c3">Thursday</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">perform a heat shock transformation of psb1a7 -- this could not be done on Wed. because no plates were left to perform this. Had to make plates first.</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">Add 1uL of resuspended</span><span class="c2 c3"> psb1A7</span><span class="c2"> in 10uL of competent cells (Novablue). (thawed on ice)</span></li></ol><p class="c4 c13"><span class="c2 c11 c3">See Protocols page for Heat Shock Transformation </span></p><p class="c9"><span class="c2">&nbsp;</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Mini-prep of psB1A3, send sequence w/ RFP F,R</span></li><li class="c6"><span class="c2">Gel-purify psb1A3 pcr, send off to sequencing w/ RFP F,R</span></li></ol><p class="c4"><span class="c0 c3">Friday</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Start liquid culture of psB1A7 </span></li></ol><ol class="c1"><li class="c6" value="1"><span class="c2">&nbsp;</span></li><li class="c6"><span class="c2">Digest - Debika (12-2 pm)</span></li><li class="c6"><span class="c2">Ligate, transform - Mitesh, Scott (5pm)</span></li></ol><p class="c4"><span class="c0 c3">Saturday</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Colony PCR of constructs (10 each)</span></li><li class="c6"><span class="c2">Mini-prep of psb1A7- Scott (9am)</span></li><li class="c6"><span class="c2">Run gel</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">hybB F, AOX R</span></li><li class="c8"><span class="c2">hybB F, ompA R</span></li><li class="c8"><span class="c2">ompA F, AOX R</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Need controls for everything from now on!</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Controls: (Can&rsquo;t read Scott&rsquo;s handwriting, sorry Scott! This stuff is still on the whiteboard in the lab)</span></p><p class="c4"><span class="c2">Ligate </span></p><ol class="c1"><li class="c6" value="1"><span class="c2">leave out a piece of the ligation so we know it won&rsquo;t work (- control)</span></li><li class="c6"><span class="c2">continue to use this throughout the transformation</span></li></ol><p class="c4"><span class="c2">Transformation control</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Just digest psB1A7 use it to transform (negative control</span></li></ol><hr><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">10/7/2010 (</span><span class="c0 c3">Thursday) </span></p><p class="c4"><span class="c2">GOALS:</span></p><p class="c4"><span class="c2">&nbsp;</span></p><ol class="c1"><li class="c6" value="2"><span class="c2">perform a heat shock transformation of psb1a7 -- this could not be done on Wed. because no plates were left to perform this. Had to make plates first. (completed Christina, Rob)</span></li></ol><ol class="c1"><li class="c6" value="3"><span class="c2">Mini-prep of psB1A3 (completed, Christina, Rob)</span></li><li class="c6"><span class="c2">send sequence w/ RFP F,R </span></li><li class="c6"><span class="c2">Gel-purify psb1A3 pcr, send off to sequencing w/ RFP F,R </span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">Make gel in Hammer Lab Chamber (the orange one) (gel completed, christina)</span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">Gel tray is max 80 or so ml </span></li><li class="c10"><span class="c2">perform gel extraction (I can do this in afternoon, Scott)</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">Results and Observations from plates made on 10-6-10: </span></p><ol class="c1"><li class="c6" value="1"><span class="c2">psb1a3 had growth; no pink colonies (may be slight, can ask Ryan as she is expert)</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">made a starter culture from these plates and see if they turn pink. </span></li></ol><ol class="c1"><li class="c6" value="2"><span class="c2">psb1a7 had no growth - plates thrown out. </span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Protocols:</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Moved the plates of the psb1a7, psb1a3 transformations to fridge -- psb1A7 plates thrown out. (Scott)</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Make starter cultures from psb1A3/NB plates made on 10/6/10.(Christina)</span></p><p class="c4"><span class="c0 c3">&nbsp;</span></p><p class="c4"><span class="c0">perform a heat shock transformation of psb1a7 -- this could not be done on Wed. because no plates were left to perform this. Had to make plates first (Christina)</span></p><p class="c4 c13"><span class="c2">Add 1uL of resuspended</span><span class="c2 c3"> psb1A7</span><span class="c2"> in 10uL of competent cells (Novablue). (thawed on ice)</span></p><p class="c4 c13"><span class="c2 c11 c3">See Protocols page for Heat Shock Transformation </span></p><p class="c4 c13"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Mini-prep of psB1A3 from starter cultures grown on 9-6-10 (Christina)</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Autoclaved a variety of pipette tip boxes to stock up(Christina)</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">make a new 1% gel in the orange pcr chamber (the chamber is the casting tray -- GENIUS!) (Christina)</span></p><p class="c4"><span class="c2 c11 c3">See Protocols page for Preparing 1% Agarose Gels</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">PCR purifcation of psb1a3 pcr wiith RFP-F, RFP-R</span></p><p class="c4"><span class="c2 c11 c3">See Protocols page for PCR Purifacation</span></p><p class="c4"><span class="c0">&nbsp;</span></p><p class="c4"><span class="c0">Gel extraction of PCR from psb1a3 using primers RFP-F, RFP-R (Scott, Gita)</span></p><p class="c4"><span class="c2 c11 c3">See Protocols page for Gel Extraction</span></p><p class="c4"><span class="c2">Note: To elute DNA, added 30 &nbsp;&mu;L water (pH 7.0 &ndash; 8.5), let the column stand for 1 min, and then centrifuged for 1 min.</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">RE Digest Recipe for RFP-F3R (that Debika did this yesterday)(Christian is doing the digest today)</span></p><p class="c4"><span class="c2">12.5 uL H20 </span></p><p class="c4"><span class="c2">5uL 10X Promega Buffer B</span></p><p class="c4"><span class="c2">6 uL RFP-F3R (167.4 ng/uL 10.6.2010)</span></p><p class="c4"><span class="c2">5 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c4"><span class="c2">0.75 &nbsp;uL SpeI</span></p><p class="c4"><span class="c2">0.75 &nbsp;uL XmaI</span></p><p class="c4"><span class="c2 c3">Total=50 ul total</span></p><p class="c4"><span class="c2">Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)</span></p><p class="c4"><span class="c2">Start 5pm</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">10/8/2010</span></p><p class="c4"><span class="c0">Goals</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Nanospec the gel extract done on 10/7/2010 (of psb1a3 RFP-FR, 700 bp band)</span></li><li class="c6"><span class="c2">Run 4 uL of the gel extract on the gel and take picture (to prove psb1a3 has rfp!)</span></li><li class="c6"><span class="c2">Send off for sequencing:</span></li></ol><ol class=""><li class="c8" value="1"><span class="c2">gel extract with RFP FR</span></li><li class="c8"><span class="c2">the mini prep of psb1a3 with RFP FR (mini prep from thursday)</span></li></ol><ol class="c1"><li class="c6" value="4"><span class="c2">Repeat transformation of psb1a7!</span></li><li class="c6"><span class="c2">PCR purification of digest from (10/7/2010) of RFP F3R</span></li></ol><p class="c4 c13"><span class="c2 c11 c3">See Protocols page for PCR Purification</span></p><ol class="c5"><li class="c8" value="1"><span class="c2">run on gel to check for 700 bp band</span></li><li class="c8"><span class="c2">if ok, then </span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">ligate to Hyb.ompa and psb1a3 or psb1a7 (which vector)?</span></li></ol><p class="c4"><span class="c0">Observations</span></p><p class="c4"><span class="c2">The transformation of psb1a7 into NB did not work. We should check whether the strain is compatible with the plasmid. &nbsp;</span></p><p class="c4"><span class="c2">The colonies from the transformation of psb1a3 directly from well into NB have turned slightly pink. </span></p><p class="c4"><span class="c2">Liquid cultures of psb1a3 colonies from plates from 10.6.2010 appear brown with no pink. However, when we pelleted them for miniprep, the pellet was pink!</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">minprep of psb1a3 liq culture from palte from10.6.2010</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Transformation of pSB1A7 in NB cells (with + control)</span></p><p class="c4"><span class="c2">Megan suggested we use fresh NB cells (from Gaucher cells), use a positive control (known good plasmids)</span></p><p class="c4"><span class="c2 c11">2 reactions:</span></p><p class="c4"><span class="c2">10 &#1405;L Nova Blue cells + 5 &#1405;L of mRFP from Gaucher Lab (labeled mRFP mini prep 3/11/09)</span></p><p class="c4"><span class="c2">10 &#1405;L Nova Blue cells + 2&#1405;L of psb1a7 from &nbsp;well aliquot</span></p><p class="c4"><span class="c2"> </span></p><p class="c4"><span class="c2 c11 c3">See Protocols page for Heat Shock Transformation.</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">PCR purification of digest from (10/7/2010) of RFP F3R (Gita)</span></p><p class="c4"><span class="c2 c11 c3">See Protocols page for PCR Purification.</span></p><p class="c4"><span class="c2">notes: elute in 30 uL!</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Nanospec</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">purified digest of RFP-F3R (labeled with 10.8.2010) = 18.1 ng/uL</span></li><li class="c6"><span class="c2">Gel extract of RFP FR (tube labeled 1, date 10.7.2010) = 84 ng/uL</span></li><li class="c6"><span class="c2">Gel extract of RFP FR (tube labeled 2, date 10.7.2010) = 91.3 ng/uL</span></li><li class="c6"><span class="c2">psb1a3 mini prep </span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">tube 1: 64 ng/uL</span></li><li class="c8"><span class="c2">tube 2 :56 ng/uL</span></li></ol><p class="c4"><span class="c0">&nbsp;</span></p><p class="c4"><span class="c0">Run on 1% gel</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Gel extractions from 10.7.2010 of psb1a3+ RFP-FR (2 tubes)</span></li><li class="c6"><span class="c2">purified, digested RFP-F3R from today, 10.8.2010</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">For tomorrow</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">high priority</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">pick colonies of psb1a7 (if succesful) and transfer to liquid culture</span></li><li class="c8"><span class="c2">tranform cells using psb1c3, since it is the vector we have to submit in. </span></li><li class="c8"><span class="c2">ligate RFP F3R to hyb.ompa and psb1a7 (likely have to be done sunday)</span></li></ol><ol class="c1"><li class="c6" value="2"><span class="c2">autoclave more water</span></li><li class="c6"><span class="c2">Find some chloramphenicol since we have to submit parts in psb1c3 and it has chloramphenicol resistance</span></li><li class="c6"><span class="c2">low priority</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">run pcr on psb1a3 minipreps today to check for rfp</span></li></ol><p class="c4 c13"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c2 c3">10/9/2010</span></p><p class="c4"><span class="c0">Goals</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Troubleshoot psb1a7, psb1a3</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">List of wells, inserts, and sequences (</span><span class="c0"><a href="http://www.google.com/url?q=http%3A%2F%2Fpartsregistry.org%2Fassembly%2Flibraries.cgi%3Fid%3D31&amp;sa=D&amp;sntz=1&amp;usg=AFQjCNG0fXu0QjGrIZuxeCNGIxGvHiHArw">http://partsregistry.org/assembly/libraries.cgi?id=31</a></span><span class="c2">)</span></li></ol><p class="c4"><span class="c0">Notes</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">Troubleshoot psb1a7</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">does it have a cbbd toxic gene in it?</span></li></ol><ol class=""><li class="c10" value="1"><span class="c2">No, it has an insert coding for a antibody chain, part: K126000</span></li></ol><p class="c4 c15"><span class="c2">(</span><span class="c0"><a href="http://www.google.com/url?q=http%3A%2F%2Fpartsregistry.org%2Fwiki%2Findex.php%3Ftitle%3DPart%3ABBa_K126000&amp;sa=D&amp;sntz=1&amp;usg=AFQjCNFBJXz_hFsVsi4FZU0lrYCcPps-HA">http://partsregistry.org/wiki/index.php?title=Part:BBa_K126000</a></span><span class="c2">) </span><span class="c0"><a href="">(http://partsregistry.org/assembly/plates.cgi?id=1259)</a></span></p><p class="c4"><span class="c2">&nbsp;</span></p><ol class="c5"><li class="c8" value="2"><span class="c2">can we digest (is it digested) and ligate it directly from the well to our parts?</span></li></ol><ol class="c12"><li class="c10" value="1"><span class="c2">should we pcr it first?</span></li></ol><ol class="c5"><li class="c8" value="3"><span class="c2">do we have a ccdb tolerant strain?</span></li></ol><p class="c4 c13"><span class="c2">&nbsp;</span></p><ol class="c1"><li class="c6" value="2"><span class="c2">Trobuleshoot psb1a3</span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">psb1a3 has RFP </span><span class="c0"><a href="">(http://partsregistry.org/partsdb/get_part.cgi?part=pSB1A3)</a></span><span class="c2"> </span></li><li class="c8"><span class="c2">BBa_J04450 = RFP</span></li></ol><ol class="c1"><li class="c6" value="3"><span class="c2">tranform cells using psb1c3, since it is the vector we have to submit in. </span></li></ol><ol class="c5"><li class="c8" value="1"><span class="c2">Have plasmid backbone from kit (Where is it?)</span></li><li class="c8"><span class="c2">Has RFP insert for in well Plate 1 - A3 (</span><span class="c0"><a href="http://www.google.com/url?q=http%3A%2F%2Fpartsregistry.org%2Fassembly%2Fplates.cgi%3Fid%3D1257&amp;sa=D&amp;sntz=1&amp;usg=AFQjCNGZEL21q96T5macGA-sc1m3waRKlQ">http://partsregistry.org/assembly/plates.cgi?id=1257</a></span><span class="c2">)</span></li></ol><p class="c4"><span class="c2">&nbsp;</span></p><p class="c4"><span class="c0">Protocols</span></p><p class="c4"><span class="c2"></span><span class="c0">RE Double Digest Recipe for pSB1A3 (from 10/8/2010)</span></p><p class="c4"><span class="c2">6.5 uL H20 </span></p><p class="c4"><span class="c2">3uL 10X Promega Buffer E </span></p><p class="c4"><span class="c2">16 uL pSB1A3 (10.8.2010, 64 ng/uL)(tube 1)</span></p><p class="c4"><span class="c2">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c4"><span class="c2">0.75 uL SpeI</span></p><p class="c4"><span class="c2">0.75 uL EcoRI</span></p><p class="c4"><span class="c2 c3">Total=30 ul total</span></p><p class="c4"><span class="c2">Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)</span></p><p class="c4"><span class="c2">Predict a insert (1000 bp) and plasmid (2000 bp)</span></p><p class="c4"><span class="c0">RE Digest Recipe for pSB1A3 (from 10/8/2010)</span></p><p class="c4"><span class="c2">5.0 uL H20 </span></p><p class="c4"><span class="c2">2.5 uL 10X Promega Buffer E </span></p><p class="c4"><span class="c2">16 uL pSB1A3 (10.8.2010, 50 ng/uL)(tube 2)</span></p><p class="c4"><span class="c2">3 uL BSA (1ug/uL, to a final conc of .1mg/ml)</span></p><p class="c4"><span class="c2">0.75 uL SpeI</span></p><p class="c4"><span class="c2 c3">Total=30 ul total</span></p><p class="c4"><span class="c2">Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)</span></p><p class="c4"><span class="c2">Digest start::12pm</span></p><p class="c4"><span class="c0">For Tomorrow:</span></p><ol class="c1"><li class="c6" value="1"><span class="c2">PREFORM PCR on WHITE colonies to check for what is in them: triple ligation plates from Oct </span></li><li class="c6"><span class="c2">Ask Gaucher lab if they have 1) ccdb resistance strains 2) vector lacking promoters and have amp resistance 3) the three tubes of plasmid backbone from the standard iGem kit</span></li><li class="c6"><span class="c2">Need more Promega Buffer E</span></li></ol><p class="c4"><span class="c2 c11">&nbsp;</span></p>

Latest revision as of 02:56, 28 October 2010

Georgia Institute of Technology iGEM Team 2010 Homepage

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10/3-10/9

10/3/10

For Today:

  1. Run on a gel
  1. aoxa, aoxb next to the troubleshooting pcrs from today, specifically of the PCRS using Ompa-F and Aoxa/b-R since they are similar sizes
  1. Take out plates from incubator. Record observations
  1. Run colony PCRs on at least 20 colonies per construct (yes 20)
  1. Check which primers to use for colony pcr! We can likely use the primers we already have
  2. Run results on gel

 

Results: plated colonies from 10/2/10 (Mitesh & Rob):

Observed that HybB-ompA-Aox1(a/b) constructs were successfully transformed- strong colony growth and red colonies (strange- given that these were supposedly constructs without RFP). The color strongly suggests RFP. Red colony patterns show no consistent trends- some are densely red in the middle of the plate, others mainly around the edges- red intensity varied widely even on the same plate.

 

 

Colony PCR of HybB-ompA-Aox constructs

Picked colonies from plates of HybB-ompA-Aox-RFP and added to 25 ul of water each,10 colonies from Aox1a containing plates, and 10 from Aox1b containing plates:

 

HybB-ompA-Aox1a-RFP: 1.5 mL Tubes 1-10

HybB-ompA-Aox1b-RFP: 1.5 mL Tubes 11-20

 

Used 5 ul for PCR, and added 250 ul of LB media to the rest.

 

Colony PCRs (for each colony): (Rob, Mitesh & Scott)

5 uL of cells

25.5 uL H2O

10 uL GOTAQ 5X Reaction buffer

5 uL HybB-F forward primer

5 uL RFP-R reverse primer

1 uL dNTP 10 mM - (thawed & kept on ice)

0.5 uL polymerase enzyme, TAQ

Total Volume= 50 uL

 

Colony PCRs (for each colony):

5 uL of cells

25.5 uL H2O

10 uL GOTAQ 5X Reaction buffer

5 uL HybB-F forward primer

5 uL Aox1a or b-R reverse primer

1 uL dNTP 10 mM - (thawed & kept on ice)

0.5 uL polymerase enzyme, TAQ

Total Volume= 50 uL

Important: For the PCR tubes, the ones labeled 1,3,5,7,9 are treated with Aox1a reverse primer.

All the even number (2,4,6,...16,18,20) labeled have RFP-R reverse primer in them. And the tubes labeled 11,13,15,17,19 have Aox 1bR reverse primer in them.

PCR start time: 6 PM

 

For Monday:

  1. Check colony pcr on a gel
  1. calculate the predicted sizes and compare to gel results
  1. Continue with colony PCRS if more info is required
  2. Check triple ligations on gel

 

10/4/2010

Protocols

 

1 % Gel to Check colony PCRs from 10/3/2010

Started at 9 am

Order:ladder:1-6|ladder|7-12|ladder|13-14

Important: For the PCR tubes, the ones labeled 1,3,5,7,9 are treated with Aox1a reverse primer.

All the even number (2,4,6,...16,18,20) labeled have RFP-R reverse primer in them. And the tubes labeled 11,13,15,17,19 have Aox 1bR reverse primer in them.

PCR start time: 6 PM

gel start at 10:34 am

 

Predicted Bands:

Hyb-F + RFP-R approx 2100-2200  bp

Hyb-F + Aoxa-R - approx 1500

Hyb-F + Aoxb-R approx 1500

 

Observations- 2200 bp band is seen, but other bands are also observed for the Hyb-F and RFP-R reactions

 

Suggestions:

  1. Troubleshoot by PCR sequencing the basic products (e.g. sequence just HybB in one tube, in the next, just OmpA; in the next, just AOX; in the next, just RFP). This will tell us whether each gene is actually present in the cells PCRed.
  2. Start over again entirely, from the very beginning, digesting each individual gene, ligating, transforming.
  3. Start using controls. When possible, include positive and negative controls.
  1. For example, when doing a double-ligation, for instance, perform experiment alongside designed to fail (e.g. lacking one of the ligation genes) for a negative control.
  2. To see if the plasmid pSB1A3 is causing our problems, transformed this plasmid alone directly into E. coli and plate.
  1. If pSB1A3 turns out to be the problem, can attempt to use a different plasmid backbone. We’ve researched and found plasmid pSB1C3 to have the desired characteristics, including lack of a  promoter, beginning EcoRI and ending SpeI restriction sites.

 

Plan to go forward:

1. Transform “plain” plasmid pSB1A3 into E. coli today 4 OCT 2010. Check for RFP expression.

2. In tandem with step 3, troubleshoot by sequencing the basic gene products in the “bad” HybB-OmpA-AOX contsructs, looking specifically for each gene.

3. In tandem with step 2, re-do the entire construction process from the individual genes up.

4. Transform cells using new biobrick vectors, like psb1c3, and use those instead of psb1a3!

 

NOTE: Need to make more media plates!

 

Protocols

 

Transformation of NB cells using pSB1a3

10 սL Nova Blue cells + 5 սL of pSB1A3 from MiniPrep

 

1. Left cells and plasmid on ice.

2. Added plasmid (5սL) to cells. Mix gently by swirling pipette tip in mixture (DO NOT ASPIRATE).

3. Left cells on ice for 30 min.

4. Applied heat shock of 45 seconds in 42C bath.

5. Put tubes on ice for 2 min.

6. Added 250 սL of LB (room temp.)

7. Incubated 1 hour at 37 C

8. Plated 100սL (x2 plates) and left plates in the 37 degrees incubator  

9. Incubated overnight at 37C.

 

Troubleshooting PCRs

For each test (e.g. HybB, OmpA, AOX1a, etc.)

2 uL of cells

9.4 uL H2O

4 uL GOTAQ 5X Reaction buffer

2 uL forward primer

2 uL reverse primer

0.4 uL dNTP 10 mM - (thawed & kept on ice)

0.2 uL polymerase enzyme, GOTAQ

Total Volume= 20 uL  

 

Combinations

A: HybB-OmpA-AOX1a-pSB1A3 in NovaBlue cells, ligated 2 OCT 2010 (Margo + Scott)

Selected 1 red colony, suspended in 20 uL sterile dH2O

1: HybB forward & reverse primers

2: OmpA forward & reverse primers

3: AOX1a forward & reverse primers

4: RFP forward & reverse primers

 

Selected 1 white colony, suspended in 20 uL sterile dH2O

5: HybB forward & reverse primers

6: OmpA forward & reverse primers

7: AOX1a forward & reverse primers

8: RFP forward & reverse primers

 

B: HybB-OmpA-AOX1b-pSB1A3 in NovaBlue cells, ligated 2 OCT 2010 (Margo + Scott)

Selected 1 red colony, suspended in 20 uL sterile dH2O

1: HybB forward & reverse primers

2: OmpA forward & reverse primers

3: AOX1a forward & reverse primers

4: RFP forward & reverse primers

 

Selected 1 white colony, suspended in 20 uL sterile dH2O

5: HybB forward & reverse primers

6: OmpA forward & reverse primers

7: AOX1a forward & reverse primers

8: RFP forward & reverse primers

 

P: “plain” mini-prepped pSB1A3 plasmid

        RFP forward & reverse primers

 

Ran a PCR on the samples using 1KB ladder -- ran A 1-7

Lane         1: ladder

2: A1

3: A2

4: A3

5: A4

6: A5

7: A6

8: A7

 

 

Note: need to make a big batch of 1% gels tomorrow since we are running so many samples.

Need more agarose from Gaucher Lab. COMPLETED

Need more EtBr. COMPLETED

Need a 100bp Ladder to see some of this stuff (small constructs) -- we might need to get Ryan to order some.

 

10-5-10

Obtain some more agarose and EtBr from Gaucher lab.

Make 1% gels.

See Protocols page for Preparing 1% Agarose Gels

Ran a gel on the remaining PCR samples from 10-4-10

Following 1 KB ladder, lanes should read in order:

A8, P, B1, B2, B3, B4, B5, B6, B7, B8.

No digital picture available, but one was printed and is in the lab, labelled with the date.

 

  1. Starting construct building strategy over again
  1. Reactions:
  1. HyBb F,R (template biobrick)
  2. OmpA F,R (template ompa vector)
  3. Aox1a F,R (template synthesized gene)
  4. Aox1a F,R2 (template synthesized gene)
  5. Aox1b F,R (template synthesized gene)
  6. Aox1b F,R2 (template synthesized gene)
  7. RFP F,R (template RFP plasmid) (from 9.27.2010 miniprep)
  8. RFP F2,R  (template RFP plasmid) (from 9.27.2010 miniprep)

 

PCR Protocol (Scott)

  1. 26.5 uL H2O
  2. 10 uL PHUSION 5X Reaction buffer
  3. 5 uL forward primer
  4. 5 uL reverse primer
  5. 1 uL dNTP 10 mM - (thawed & kept on ice)
  6. 2 uL template DNA
  7. 0.5 uL polymerase enzyme, PHUSION

Total Volume= 50 uL

started 4:33pm

 

10/6/2010

Goals

  1. The gel from 10/5/2010 of the troubleshooting pcrs, specifically the reaction of pSB1A3 with RFP-F and R, suggests that the vector used for ligation has RFP. We will use anothe rvector from biobrick
  2. We want to transform cells with the new biobrick vector today, miniprep the cells tomorrow morning, digest, and ligate to our hyb.ompa+aox that we have or are making now. Finish by friday is the goal.
  3. Sequence hyb-RFP (for bronze medal requirements)
  4. begin hyb-ompa-rfp building when primers come in
  5. I saw Richard’s sequence was for psb1c3, but we are using psb1a3; this may a primer and digests! Investigate!
  6. Sequence white colonies from the plates on 10/3/2010
  7. For today:
  1. Reconstitute new biobrick vector (psb1A7) COMPLETED
  2. Transform NB cells using new biobrick vector COMPLETED
  3. Check PCRs from 10/5/2010 on gel (tubes in yellow pcr box labeled 1-8 and dated 10.5) COMPLETED
  4. Make Amp plates
  5. Autoclave pipette tips
  1. This involves testing which tips are compatible with ours (we have lots of random tips)

 

Notes on Vector-

Here is an experience from another team concerning transcription of a product during an “off state”:

 

Our team (Davidson College) has used this vector in E. coli strain JM109 extensively. Clones are Amp resistant and are recovered in reasonable yields from mini preps. One drawback is that the vector has read-through transcription that comes into the cloned BioBrick part from both directions. This problem is a major one when an "off state" is critical for the control of a device. Parts without a promoter are expressed in this vector, regardless of orientation (see pSB1A7 Part Design for details). Reverse read-through might be from the Amp resistance gene. Forward read-through might be from a cryptic promoter, since no known genes in the backbone are in the plus orientation.

--Kahaynes 15:48, 23 October 2006 (EDT)

 

Alternative vectors:

  1. pSB1A7 (2010 Kit plate 3, 15G well, name BBa_K126000)
  1. Reasons:
  1. There are double terminators on either side of the multiple cloning site that block any read through or unwanted transcription from the plasmid promoters (plasmid promoters are involved in antibiotic production)
  2. Ampicillin resistance
  3. Already in kit

 

 

Protocols

1. Resuspend psb1A7 DNA in 10uL of autoclaved water.

 

 

3. Run gels of PCR’s on 10-5-10.

 

Order:

  1. HyBb F,R (template biobrick)
  2. OmpA F,R (template ompa vector)
  3. Aox1a F,R (template synthesized gene)
  4. Aox1a F,R2 (template synthesized gene)
  5. Aox1b F,R (template synthesized gene)
  6. Aox1b F,R2 (template synthesized gene)
  7. RFP F,R (template RFP plasmid) (from 9.27.2010 miniprep)
  8. RFP F2,R  (template RFP plasmid) (from 9.27.2010 miniprep)

 

Growing colony of pSB1a3 transformed cells (Scott)

I picked 3 colonies from the plate marked “MFC 9.15.2010 C1 “ from 9.15.2010, with the psb1a3 transformed cells that are slighty pink. I did 3 mL + 3 uL of Carb, and put them in the incubator.

 

PCR of pSB1A3 (direct from well aliquot) using RFP-F,R

  1. 27.5 uL H2O
  2. 10 uL GoTaq 5X Reaction buffer
  3. 5 uL forward primer (RFP-F)
  4. 5 uL reverse primer (RFP-R)
  5. 1 uL dNTP 10 mM - (thawed & kept on ice)
  6. 1 uL template DNA
  7. 0.5 uL polymerase enzyme, GoTaq

Total Volume= 50 uL

Left in block B- Thursday people need to retieve it- Need to gel purify- scroll down to see a lit of things to do for Thursday. These steps are also highligted in blue below.

PCR of mRFP miniprep (direct from well aliquot) using RFP-F3,R

  1. 26.5 uL H2O
  2. 10 uL GoTaq 5X Reaction buffer
  3. 5 uL forward primer (RFP-F3)
  4. 5 uL reverse primer (RFP-R)
  5. 1 uL dNTP 10 mM - (thawed & kept on ice)
  6. 2 uL template DNA
  7. 0.5 uL polymerase enzyme, GoTaq

Total Volume= 50 uL

Left in block B- Thursday people need to retieve it  - Scott knows what to do with this. I don’t. -Debika

 

 

Transformation of NB using pSB1A3 direct from Gel aliquot) -completed (Christina, Debika)

10 սL Nova Blue cells + 5 սL of pSB1A3 from MiniPrep

 

1. Left cells and plasmid on ice.(Scott)

2. Added plasmid (5սL) to cells. Mix gently by swirling pipette tip in mixture (DO NOT ASPIRATE).

3. Left cells on ice for 30 min.

4. Applied heat shock of 45 seconds in 42C bath. (Debika)

5. Put tubes on ice for 2 min. (I messed up and put in LB in a minute instead of 2 minutes. Left it in ice again for a minute after adding LB). Don’t know how that will affect our results. Sorry if this transformation doesn’t work).

6. Added 250 սL of LB (room temp.)

7. Incubated 1 hour at 37 C

8. Plated 100սL (x2 plates) and left plates in the 37 degrees incubator. (Christina)  

9. Incubated overnight at 37C.  

 

Transformation of NB using pSB1A7 direct from Gel aliquot) -completed (Christina)

10 սL Nova Blue cells + 5 սL of pSB1A3 from MiniPrep

  1. Add 1uL of resuspended psb1A7 in 10uL of competent cells (Novablue). (thawed on ice)

See Protocols page for Heat Shock Transformation

 

Note: The incubation step was skipped, which likely will lead to little growth


 

Meeting Notes

Priority List

Critical

  1. hyb.ompa.aox construct (in pSB1A7)
  2. hyb.ompa.RFP construct (in pSB1A7) (Scott)
  3. Sequencing of constructs and conformation with standard
  4. Biobrick

Need

  1. Calorimetric/Heat  (Rob, Christian)
  2. Periplasmic Expression Picture (Fluorescent Microscope) (Scott)
  3. Cold Shock Quantification (hybB)
  4. Modeling (Gita, Mitesh)
  1. Liquid culture
  2. Solid Media

        Want

  1. Confirm pSB1A3 has RFP in it
  1. Pick colony from plates, liquid culture. (COMPLETED)(SCOTT)  “MFC 9.15.2010 C1 “

Thursday: Mini-prep, off to sequencing. Ask Ryan or Megan

    ii     Aliquot from well, PCR (COMPLETED, DEBIKA, left in right side block overnight)

Thursday: gel purify, off to sequencing. Ask Ryan or Megan


 

Detailed Plan

Today (wed)

Critical Actions

  1. hyb.ompa.rfp construct
  1. Start PCR of RFP using new primer using a plasmid with ryan’s rfp (pcold and rfp, or the gaucher lab rfp plasmid) (Debika, completed)

 

Want Actions

  1. confirm PSB1A3 has RFP
  1. Scott
  1. Pick colony from original psb1a3 plates  “MFC 9.15.2010 C1 “ and put in liq culture (completed)
  1. Debika
  1. Start PCR of psb1a3 directly from well, using primers RFP-F and RFP-R (Debika, completed)
  1. Christina, Debika,
  1. transformation of NB cells using original psb1a3 aliquot (completed)

 

Other Actions

  1. order psb1a7 sequencing primers (the forward psb1a3-F primer is ok, but we need a reverse primer)

 

Thursday

  1. perform a heat shock transformation of psb1a7 -- this could not be done on Wed. because no plates were left to perform this. Had to make plates first.
  1. Add 1uL of resuspended psb1A7 in 10uL of competent cells (Novablue). (thawed on ice)

See Protocols page for Heat Shock Transformation

 

  1. Mini-prep of psB1A3, send sequence w/ RFP F,R
  2. Gel-purify psb1A3 pcr, send off to sequencing w/ RFP F,R

Friday

  1. Start liquid culture of psB1A7
  1.  
  2. Digest - Debika (12-2 pm)
  3. Ligate, transform - Mitesh, Scott (5pm)

Saturday

  1. Colony PCR of constructs (10 each)
  2. Mini-prep of psb1A7- Scott (9am)
  3. Run gel
  1. hybB F, AOX R
  2. hybB F, ompA R
  3. ompA F, AOX R

 

Need controls for everything from now on!

 

Controls: (Can’t read Scott’s handwriting, sorry Scott! This stuff is still on the whiteboard in the lab)

Ligate

  1. leave out a piece of the ligation so we know it won’t work (- control)
  2. continue to use this throughout the transformation

Transformation control

  1. Just digest psB1A7 use it to transform (negative control

 

10/7/2010 (Thursday)

GOALS:

 

  1. perform a heat shock transformation of psb1a7 -- this could not be done on Wed. because no plates were left to perform this. Had to make plates first. (completed Christina, Rob)
  1. Mini-prep of psB1A3 (completed, Christina, Rob)
  2. send sequence w/ RFP F,R
  3. Gel-purify psb1A3 pcr, send off to sequencing w/ RFP F,R
  1. Make gel in Hammer Lab Chamber (the orange one) (gel completed, christina)
  1. Gel tray is max 80 or so ml
  2. perform gel extraction (I can do this in afternoon, Scott)

 

Results and Observations from plates made on 10-6-10:

  1. psb1a3 had growth; no pink colonies (may be slight, can ask Ryan as she is expert)
  1. made a starter culture from these plates and see if they turn pink.
  1. psb1a7 had no growth - plates thrown out.

 

Protocols:

 

Moved the plates of the psb1a7, psb1a3 transformations to fridge -- psb1A7 plates thrown out. (Scott)

 

Make starter cultures from psb1A3/NB plates made on 10/6/10.(Christina)

 

perform a heat shock transformation of psb1a7 -- this could not be done on Wed. because no plates were left to perform this. Had to make plates first (Christina)

Add 1uL of resuspended psb1A7 in 10uL of competent cells (Novablue). (thawed on ice)

See Protocols page for Heat Shock Transformation

 

Mini-prep of psB1A3 from starter cultures grown on 9-6-10 (Christina)

 

Autoclaved a variety of pipette tip boxes to stock up(Christina)

 

make a new 1% gel in the orange pcr chamber (the chamber is the casting tray -- GENIUS!) (Christina)

See Protocols page for Preparing 1% Agarose Gels

 

PCR purifcation of psb1a3 pcr wiith RFP-F, RFP-R

See Protocols page for PCR Purifacation

 

Gel extraction of PCR from psb1a3 using primers RFP-F, RFP-R (Scott, Gita)

See Protocols page for Gel Extraction

Note: To elute DNA, added 30  μL water (pH 7.0 – 8.5), let the column stand for 1 min, and then centrifuged for 1 min.

 

RE Digest Recipe for RFP-F3R (that Debika did this yesterday)(Christian is doing the digest today)

12.5 uL H20

5uL 10X Promega Buffer B

6 uL RFP-F3R (167.4 ng/uL 10.6.2010)

5 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75  uL SpeI

0.75  uL XmaI

Total=50 ul total

Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)

Start 5pm

 

10/8/2010

Goals

  1. Nanospec the gel extract done on 10/7/2010 (of psb1a3 RFP-FR, 700 bp band)
  2. Run 4 uL of the gel extract on the gel and take picture (to prove psb1a3 has rfp!)
  3. Send off for sequencing:
  1. gel extract with RFP FR
  2. the mini prep of psb1a3 with RFP FR (mini prep from thursday)
  1. Repeat transformation of psb1a7!
  2. PCR purification of digest from (10/7/2010) of RFP F3R

See Protocols page for PCR Purification

  1. run on gel to check for 700 bp band
  2. if ok, then
  1. ligate to Hyb.ompa and psb1a3 or psb1a7 (which vector)?

Observations

The transformation of psb1a7 into NB did not work. We should check whether the strain is compatible with the plasmid.  

The colonies from the transformation of psb1a3 directly from well into NB have turned slightly pink.

Liquid cultures of psb1a3 colonies from plates from 10.6.2010 appear brown with no pink. However, when we pelleted them for miniprep, the pellet was pink!

 

minprep of psb1a3 liq culture from palte from10.6.2010

 

Transformation of pSB1A7 in NB cells (with + control)

Megan suggested we use fresh NB cells (from Gaucher cells), use a positive control (known good plasmids)

2 reactions:

10 սL Nova Blue cells + 5 սL of mRFP from Gaucher Lab (labeled mRFP mini prep 3/11/09)

10 սL Nova Blue cells + 2սL of psb1a7 from  well aliquot

See Protocols page for Heat Shock Transformation.

 

PCR purification of digest from (10/7/2010) of RFP F3R (Gita)

See Protocols page for PCR Purification.

notes: elute in 30 uL!

 

Nanospec

  1. purified digest of RFP-F3R (labeled with 10.8.2010) = 18.1 ng/uL
  2. Gel extract of RFP FR (tube labeled 1, date 10.7.2010) = 84 ng/uL
  3. Gel extract of RFP FR (tube labeled 2, date 10.7.2010) = 91.3 ng/uL
  4. psb1a3 mini prep
  1. tube 1: 64 ng/uL
  2. tube 2 :56 ng/uL

 

Run on 1% gel

  1. Gel extractions from 10.7.2010 of psb1a3+ RFP-FR (2 tubes)
  2. purified, digested RFP-F3R from today, 10.8.2010

 

 

For tomorrow

  1. high priority
  1. pick colonies of psb1a7 (if succesful) and transfer to liquid culture
  2. tranform cells using psb1c3, since it is the vector we have to submit in.
  3. ligate RFP F3R to hyb.ompa and psb1a7 (likely have to be done sunday)
  1. autoclave more water
  2. Find some chloramphenicol since we have to submit parts in psb1c3 and it has chloramphenicol resistance
  3. low priority
  1. run pcr on psb1a3 minipreps today to check for rfp

 

10/9/2010

Goals

  1. Troubleshoot psb1a7, psb1a3
  1. List of wells, inserts, and sequences (http://partsregistry.org/assembly/libraries.cgi?id=31)

Notes

  1. Troubleshoot psb1a7
  1. does it have a cbbd toxic gene in it?
  1. No, it has an insert coding for a antibody chain, part: K126000

(http://partsregistry.org/wiki/index.php?title=Part:BBa_K126000) (http://partsregistry.org/assembly/plates.cgi?id=1259)

 

  1. can we digest (is it digested) and ligate it directly from the well to our parts?
  1. should we pcr it first?
  1. do we have a ccdb tolerant strain?

 

  1. Trobuleshoot psb1a3
  1. psb1a3 has RFP (http://partsregistry.org/partsdb/get_part.cgi?part=pSB1A3)
  2. BBa_J04450 = RFP
  1. tranform cells using psb1c3, since it is the vector we have to submit in.
  1. Have plasmid backbone from kit (Where is it?)
  2. Has RFP insert for in well Plate 1 - A3 (http://partsregistry.org/assembly/plates.cgi?id=1257)

 

Protocols

RE Double Digest Recipe for pSB1A3 (from 10/8/2010)

6.5 uL H20

3uL 10X Promega Buffer E

16 uL pSB1A3 (10.8.2010, 64 ng/uL)(tube 1)

3 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

0.75 uL EcoRI

Total=30 ul total

Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)

Predict a insert (1000 bp) and plasmid (2000 bp)

RE Digest Recipe for pSB1A3 (from 10/8/2010)

5.0 uL H20

2.5 uL 10X Promega Buffer E

16 uL pSB1A3 (10.8.2010, 50 ng/uL)(tube 2)

3 uL BSA (1ug/uL, to a final conc of .1mg/ml)

0.75 uL SpeI

Total=30 ul total

Run 3 hours in the heating block in our lab (37c, put water in the heating blocks)

Digest start::12pm

For Tomorrow:

  1. PREFORM PCR on WHITE colonies to check for what is in them: triple ligation plates from Oct
  2. Ask Gaucher lab if they have 1) ccdb resistance strains 2) vector lacking promoters and have amp resistance 3) the three tubes of plasmid backbone from the standard iGem kit
  3. Need more Promega Buffer E