Team:Colombia/Journal

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{{Http://2014.igem.org/Team:Colombia Uniandes/Bootstrap}}
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{{Http://2014.igem.org/Team:Colombia}}
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<html>
<html>
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<div class="container">
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<div class="span11" style="text-align: justify;">
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    <div class="span11">
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<h2>Journal</h2>
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<div class="tabbable tabs-left"> <!-- Only required for left/right tabs -->
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<br><br>
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  <ul class="nav nav-tabs">
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<center><b><h1  class="curs1">Journal</h1></b></center>
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    <li class="active"><a href="#tab1" data-toggle="tab">Chimi's Journal</a></li>
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<br><br>
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    <li><a href="#tab2" data-toggle="tab">Nicko's Journal</a></li>
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  </ul>
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  <div class="tab-content">
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    <div class="tab-pane active" id="tab1"><p></html>
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=='''Hands at work!'''==
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<p align="justify">
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Here you will find the overall progression of our work at the laboratory designing Chimi.
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</p>
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===='''13th June 2013'''====
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<p align="justify">
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The first thing we did was to extract the plasmids from the iGEM plaque.
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</p>
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<ol>
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<li>From the 2013 kit, Plate 1, Well 19 – o</li>
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<li>From the 2013 kit, Plate 1, Well 2 – i</li>
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<li>From the 2013 kit, Plate 3, Well 17 – c</li>
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</ol>
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The iGEM parts were taken in order to perform an electroporation. For this, we use:
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<ul>
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<li>20 µ of miliQ water (ultra pure). Find the plate and stick the tip with water into the well, perforating the aluminum.</li>
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<li>Resuspend the well’s content by gentle pipetting.</li>
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<li>When the water has a dark red color, transfer it to a PCR eppendorf and put on ice.</li>
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</ul>
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==== '''15th June 2013''' ====
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We picked the transformant colonies.
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===='''18th June 2013'''====
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<p>We performed miniprep procedures with the GenElute HP Plasmid Miniprep kit.</p>
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<p>This are the overall steps:</p>
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<ol>
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<li>Harvest cells.</li>
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<li>Resuspend cells.</li>
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<li>Cell lysis.</li>
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<li>Neutralization.</li>
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Spin method:
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<li>Prepare column.</li>
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<li>Load cleared lysate.</li>
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<li>Wash column with wash solution 1.</li>
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<li>Was column with wash solution 2.</li>
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<li>Centrifuge.</li>
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<li>Elute DNA.</li>
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</ol>
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We did a confirmation Gel 100 V x 30 min. -> It showed 1 bond in the first two wells corresponding to Nal1 and Nal2. We succesfully extracted the Nal plasmids!
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===='''June 21st, 2013'''====
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Harju et al., “Bust n’ Grab” Protocol for Yeast Genomic DNA Extraction
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<ol>
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<li>5 mL of overnight culture of ''S. cerevisiae'' (in BHI medium) were centrifuged at 8500 rpm for 5 min. Discard de supernatant.</li>
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<li>500 µL of Harju lysis buffer were added to each tube.</li>
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<li>Place 2 min at -20 °C, 1 min in water bath at 90 °C and repeat.</li>
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<li>Vortex 30 s.</li>
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<li>Add 500 µL of chloroform, vortex 2 min and centrifuge 3 min at 8500 rpm.</li>
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<li>Transfer the upper aqueous phase to a tube with 800 µL of chilled 100% ethanol and mix by inversion.</li>
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<li>Incubate for 5 min at room temperature or at 30 °C.</li>
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<li>Centrifuge for 5 min, 8500 rpm, and discard supernatant.</li>
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<li>Wash the pellet with 500 µL of ethanol (100%) by vortex. Repeat step 9.</li>
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<li>Dry pellets at room temperature or at 60 °C.</li>
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<li>Resuspend in 40 µL miliQ water.</li>
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</ol>
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===='''June 26, 2013'''====
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<ul>
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<li>We made competent yeast following the procedure mentioned before.</li>
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<li>We also made our first PCRs! We used primers 6 & 1 (A) and 34 & 9 (B) to extract VP16 and GCR from the Nal1 plasmid.</li>
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<li>Confirmation gel (2013-06-26 19 hr 16 min.jpg & 2013-06-26 19hr 15 min.jpg) with wells:
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<ol>
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<li>Ladder</li>
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<li>PCR A</li>
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<li>PCR B</li>
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<li>Miniprep for Nal. 1</li></ol></li>
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</ul>
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<p align="justify">
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A = VP16
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<br>B = GCR
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</p>
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[[File:Construct.jpg|400px|thumb|center]]
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===='''June 27, 2013'''====
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<p align="justify">
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We repeated the PCR for A and used lambda phage DNA for carrier DNA.
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We also tried extracting yeast genome using a modified Harju “Bust n’ Grab” protocol using three parallel methods:
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<ul>
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<li>Method “H” used the regular lysis buffer.</li>
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<li>Method “C” used the following lysis buffer: 3% Triton, 100 mM LiCl, 10 mM Tris-HCl, 1 mM EDTA, 100 mM NaAc.</li>
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<li>Method “O” used the following lysis buffer: 2% triton, 1& SDS, 10 mM tris-HCl, 1 mM EDTA, 100 mM LiCl.</li>
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</ul>
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</p>
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<p align="justify">
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Conformation gel was run with wells:
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<ol>
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<li>Ladder</li>
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<li>PCR A (repeated)</li>
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<li>Carrier lambda PCR</li>
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<li>Method C</li>
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<li>Method H</li>
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<li>Method O</li>
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</ol>
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The genome extraction still isn't working! :(
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</p>
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<p align="justify">
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We then ran a fusion PCR with GAL4 and VP16 (A and B). These were the PCR conditions:
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<ul>
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<li>1st PCR --> 2-step PCR
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<br>Cycle steps
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<ol>
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<li>Initial denaturation (98 °C, 30 s)</li>
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<li>15 cycles (98 °C, 10 s; 72 °C, 35 s)</li>
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<li>Final extension (72 °C, 5 min)</li>
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<li>Hold (4 °C, indefinite time)</li>
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</ol></li>
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<li>2nd PCR --> 2-step PCR (add primers)
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<br>Cycle steps
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<ol>
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<li>Initial denaturation (98 °C, 30 s)</li>
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<li>35 cycles (98 °C, 10 s; 72 °C, 35 s)</li>
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<li>Final extension (72 °C, 10 min)</li>
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<li>Hold (4 °C, indefinite time)</li>
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</ol></li>
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===='''July 2nd, 2013'''====
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Still trying to successfully extract the yeast genome! This time we tried an alternate method where we used two different solutions to break the cell wall:
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<ul>
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<li>Solution I: Glucose 50 mM, EDTA pH 8 10 mM, Tris-HCl pH 8 25 mM (esterilized) + Zymolyase</li>
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<li>Solution II: NaOH 0.2 N, SPS 1%</li>
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The rest of the protocol was taken from GenElute DNA Kit from Sigma-Aldrich.
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</ul>
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===='''July 3rd, 2013'''====
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The genome extraction was better, but it's mostly degraded DNA! We still have to improve the protocol.
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===='''July 5th, 2013'''====
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We're still improving our genome extraction protocol. This time we're trying 4 variations to break the cell wall
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<br>We're varying the incubation of both solutions, the one that comes with the kit (Proteinase K + Lysis buffer) and the zymolyase solution we previously used. The four variations were as follows.
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<ul>
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<li>A: Zymolyase for ½ h at 37 °C</li>
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<li>B: Zymolyase for ½ h at 37 °C, then protease K + lysis T ½ h at 55 °C</li>
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<li>C: Zymolyase for ½ h at 37 °C, then protease K + lysis T ½ h at 37 °C</li>
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<li>D: Regular GenElute Genome extraction protocol</li>
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</ul>
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The rest of the steps were done following the instructions from the GenElute Genome extraction protocol.
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<br>We ran a gel in this order: WM, A, B, C, D.
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<br>Both B and C gave results, with C giving better yields! We're keeping the C protocol and we're happy we can start extracting parts from the yeast genome!
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[[File:GENOMES.jpg|400px|thumb|center]]
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<br>
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<br>We performed PCRs for BAP2, GAL4, yeast terminator and pGAL1. These were the conditions:
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<br>PCR fusion 2 steps*, with process 3 at 72 °C for 45 s
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<ol>*
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<li>98 °C, 1 min</li>
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<li>98 °C, 10 s</li>
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<li>72 °C, 45 s</li>
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Steps 2 and 3 x 35
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<li>72 °C, 10 min</li>
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<li>4 °C</li>
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</ol>
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===='''July 9th, 2013'''====
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Today we ran PCRs for pGAL1 and mCherry. Now that we have several parts, we must have a clear understanding of our notation!
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===='''July 18th, 2013'''====
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Today we ran PCRs for the following fusions: F1 = TER-GCR, with primers 31 & 35; F2 = TER – mCherry, with primers 31 & 33. We also amplified E = pGAL1, using primers 13 & 15.
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===='''July 19th, 2013'''====
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Today we amplified F2 that we obtained yesterday.
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===='''July 22nd, 2013'''====
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Today we tried to fuse C-D using primers 19 and 5, TER-GCR using primers 31 and 35 and TER-mCherry using primers 31 and 33. After 15 PCR cycles, we added the primers.
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===='''July 24th, 2013'''====
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Today we extracted the terminator from the yeast genome (F1 and F2) and pGAL1 from miniprep (E). We also did PCRs to fuse again our big parts: pBAP2 – GAL4 – VP16 – GCR (CD-AB) (primers 19 & 34) and pGAL1 – mCherry (E-G) (primers 32 & 15).
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<br>At the moment, this is our progress!:
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[[File:Progress_chart.jpg|400px|thumb|center]]
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===='''August 5th, 2013'''====
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Things are advancing at a fast pace! Today we did miniprep to obtain PUC19!
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===='''August 6th, 2013'''====
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After doing a confirmation gel, C and CD are no longer with us! So we need to repeat them!
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===='''August 16th, 2013'''====
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Still no C nor D, so we did PCRs again with different methods to obtain C1, C2, D1 and D2.
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===='''August 17th,2013'''====
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We're now starting with the reporter construct's PCRs. We got the synthetised parts (E2, E3, E4 and F (the terminator)) which means we need to start fusing them. First off, we'll fuse the terminator with the reporter, since it's common to all reporters.
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===='''August 19th,2013'''====
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Today we'll perform the fusion between FG and the four Es.
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===='''August 20th,2013'''====
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The fusion didn't work out, so we reamplified FG and tried again.
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===='''August 30th, 2013'''====
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We finally have the E*GF fusions complete!
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We did the digestion for the E*GF parts and the pSB1C3 plasmid as follows:
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<ul>
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<li>Buffer 5x: 5 uL</li>
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<li>SpeI: 1 uL</li>
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<li>XbaI: 1 uL</li>
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<li>DNA: 10 uL </li>
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<li>Water: 33 uL </li>
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</ul>
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They were placed for two hours at 37°C and then were inactivated at 80°C for 20 minutes. Then we added alkaline phosphatase for 1 hour at 37°C.
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Then, we did the ligation protocol as follows:
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<ul>
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<li>Buffer 5x: 2 uL</li>
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<li>plasmid: 3 uL</li>
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<li>insert: 9 uL</li>
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<li>ligase: 1 uL</li>
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<li>Water: 5 uL</li>
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</ul>
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We then transformed them. Tomorrow we'll confirm the transformation by miniprep kit.
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===='''August 31st'''====
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After transforming them we got these minipreps
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[[File:GELminipreps.jpg|400px|thumb|center]]
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Here we can see all four E*GF parts (the four to the left of the weight marker), some with more than one plasmid conformation.
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===='''September 2nd, 2013'''====
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We contransformed the E*GF parts with the NAL plasmid in order to validate their function. Once again we did electrocompetent cells and transformed immediately.
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===='''September 3rd, 2013'''====
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We did the PCR confirmation for the parts. The bands aren't very bright but it's enough for us! We'll run the validation experiments tomorrow!
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Wells are as follows: E1FG - E1FG(2) - E2FG - E2FG(2) - Weight marker - E3FG - E3FG(2) - E4FG - E4FG(2).
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Something strange happened with E3FG(2)
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[[File:GEL7.jpg|400px|thumb|center]]
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===='''September 4th, 2013'''====
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Everything is ready for the validation experiments. We're both nervous and excited!
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We inoculated cells with dexamethasone. The original syringe had a concentration of 8mg/2mL. We obtained a total initial volume of 3.189*10^-2 µL, so we had to dilute first the dexamethasone: 9900 µL water + 100 µL dexamethasone.
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Induction experiment ON: 5 mL LB + 20 µL ampicillin + 20 µL kanamycin + 10 µL inoculum + 32 µL dexamethasone (1:1000) (inoculate ON).
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We ran a gel for several PCRs: F-H-P-E1GF2-MP-E2GF-E3GF1-E3GF2-E4GF- Everything was succesful except E1GF2.
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Then we digested the PCRs with 5 µL Buffer CutSmart + 15 µL PCR + 1 µL XbaI + 1 µL SpeI + 28 µL water (for each PCR)
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<html></p>
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    </div>
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    <div class="tab-pane" id="tab2">
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      <p></html>
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=='''Dear Journal! :)'''==
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<html>
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<p align="justify">
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Here we will place all the information about the work we have been doing during this time, it will be named by date.
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We hope you enjoy our work and thoughts as much as we do!
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</p>
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</html>
</html>
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[[File:La_foto.JPG|300px|thumb|center|Our Notebook]]
 
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===='''18-Jun-2013'''====
 
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<html>
 
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<p align="justify">
 
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Hello! Hello! Hola! Hola!
 
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After we went over the project, we proceed to design our primers so we could place our order, taking into account that we are going to use Fusion PCR as our main protocol. If you want to check our primers, go to </html>[https://2013.igem.org/Team:Colombia_Uniandes/How_to_parts How to: Parts].<html> Our primers take around 2 weeks to arrive… sooo meanwhilee all the team started making our ''E.coli'' babies electrocompetent, so we can work with them later on.
 
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</p>
 
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</html>
 
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===='''25-Jun-2013'''====
 
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<html>
 
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<p align="justify">
 
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So today we took our bacterial cells (Top 10) onto an LB medium (no antibiotics). We let it ON at 37 °C on the shaker. Also we prepared all the material we need for tomorrow: ddwater, 10 % glycerol, LB medium and microfuge tubes.
 
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</p></html>
 
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===='''26-Jun-2013'''====
 
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<html>
 
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<p align="justify">
 
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Today we are going to start preparing our cells, we inoculated in the morning and placed all our material that needs to be cold in the fridge 4 °C(ddWater and glycerol), also the centifuge rotor needs to be chilled.
 
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Then we just waited for the perfect OD and made our cells around 4:00pm. We made a lot!
 
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</p>
 
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We placed them in the -80, ready to start our work!!!
 
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</html>
 
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===='''2-Jul-2013'''====
 
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Our primers are FINALLY here and we are anxious to start working!!!
 
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So we made our plan(in theory)on big steps for the next weeks!:
 
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'''1.''' '''DNA extraction''' from ''E.coli'' and ''Cupriavidus metallidurans CH34''
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[[File:Fotojour.jpg|center|600px]]
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'''2. PCR'''
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=June 24=
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  2.1 PrcnR, RBS, rcnR, stop (Amplified as one piece)
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  2.2 PrcnA,RBS
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  2.3 hoxN, stop
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'''3.''' '''Fusion PCR'''
+
Today was the first day at the lab!! Finally! After finding out the parts we ordered from the registry had been sitting in a locker for the last two weeks and managing to salvage them we re finally up and running. First thing first, today we are making electrocompetent cells and transforming our first parts. We are using TOP 10 strain to make our transformations. We are transforming mRFP (BBa_K518012), pBAD (BBa_K206000), tetR inverter (BBa_Q04400), amilCp (BBa_K592025), pBAD-CqsA (BBa_K581011), pTet-LuxOD47E (BBa_K218017), pqrr4-GFP (BBa_K581009) and LuxU-CqsS-LuxO (BBa_K581010). We are starting with the Interlab project, so we are transforming those parts as well. For convenience, we have decided to name the Interlab parts IL1 (BBa_I20260, promoter+GFP in 1K3), IL2 (BBa_J23101, promoter), IL3 (E0240, GFP) and IL4 (BBa_J23115, promoter).
-
  3.1 PrcnA/''hoxN''
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  3.2 PrcnR,RBS,''rcnR''/PrcnA/''hoxN''
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On each PCR add 30 steps more of making gels for confirmation, again and again and again.
+
We just realized we are all out of our kanamycin stock. We need to prepare some more tomorrow.
 +
=June 25=
-
===='''3-Jul-2013'''====
+
We have transformants! Well, almost. Nothing grew on the IL4 plate :(. But nothing to worry about, we are just repeating the transformations. We made passes to liquid media of single colonies of all the other parts and we are hoping to do miniprep of all them tomorrow.
-
<html>
+
-
<p align="justify">
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Fortunately our team member Silvia Cañas already had a DNA extraction from ''E.coli'' so she donated it to us so we could start working! Thank you Silvia! :)
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We asked for ''Cupriavidus metallidurans'' to our teacher Jenny Dussan at her lab here in the university. She is giving us the strain tomorrow morning! Thank you Jenny!
+
We also cryopreserved all the bacteria we got from the registry. Just made a fresh new Kanamycin stock, so we can now transform tetR inverter.
-
This done, did the extraction of ''C.metallidurans'' and store it in the -30 °C.
+
=June 26=
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</p>
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</html>
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===='''4-Jul-2013'''====
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<html>
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<p align="justify">
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Today we started with the 2-step PCR of PrcnR, RBS, ''rcnR'', stop (Fragment that will be called "R" from now on).
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Results were negative :(
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</p>
+
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</html>
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===='''5-Jul-2013'''====
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<html>
+
-
<p align="justify">
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-
Today we repeated "R" PCR and also did ''hoxN'' with the ''Cupriavidus metallidurans'' genome. The quantities used for the reaction of ''hoxN'' are shown in the table below.
+
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Results were negative :( as we could see in our sad, SAD gel.
+
-
</p>
+
-
</html>
+
-
{| border="1" align="center"
+
-
|+'''PCR reagents and amounts for one 50 ul reaction'''
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| style="text-align: center;" |Reagent
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| style="text-align: center;" |Amount
+
-
|-
+
Our first minipreps were unsuccessful and nothing grew on the tetR inverter plate.  
-
| style="text-align: center;" |''C.metallidurans'' DNA
+
-
| style="text-align: center;" |3,0 ul
+
-
|-
+
We just realized the tetR inverter part has two other twins in the registry. We are transforming those as well and hope one of them works.
-
| style="text-align: center;" |Primer FW
+
-
| style="text-align: center;" |2,5 ul
+
-
|-
+
=June 27=
-
| style="text-align: center;" |Primer RV
+
-
| style="text-align: center;" |2,5 ul
+
-
|-
+
Today we prepared homemade lysis buffer for our minipreps, but apparently it wasn't very effective. We could not see any DNA after running our miniprerps in a gel.
-
| style="text-align: center;" |DMSO
+
-
| style="text-align: center;" |2,0 ul
+
-
|-
+
-
| style="text-align: center;" |Master Mix
+
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| style="text-align: center;" |25 ul
+
-
|-
+
-
| style="text-align: center;" |dH2O
+
-
| style="text-align: center;" |15 ul
+
-
|-
+
-
|}
+
 +
=July 1=
-
===='''9-Jul-2013'''====
+
After a long weekend, we are back in business. We have colonies in all the plates with the trnasformations of the tetR inverter! We made passes of all three parts to liquid LB for miniprep tomorrow.  
-
<html>
+
-
<p align="justify">
+
-
So, our PCRs are NOT working....We think our problem is the length of the primers and their constitution that could form secondary structures very VERY easily.
+
-
That's why we tried a new procotol of PCR for "R" with a temperature gradient, and we tried new buffers for ''hoxN''.  Reagents and quantities are shown in the table below.
+
=July 2=
-
</p>
+
-
</html>
+
-
{| border="1" align="center"
+
-
|+'''PCR reagents and amounts for one 50 ul reaction'''
+
-
| style="text-align: center;" |Reagent
+
-
| style="text-align: center;" |Amount
+
-
|-
+
We repeated the miniprep of all the parts and finally got it right this time! All molecular weights correspond to what we expected!
-
| style="text-align: center;" |''Escherichia coli'' DNA
+
-
| style="text-align: center;" |2,0 ul
+
-
|-
+
=July 3=
-
| style="text-align: center;" |Primer FW
+
-
| style="text-align: center;" |2,5 ul
+
-
|-
+
Now that we have all the parts, we can start building our construct! We are starting with the Interlab parts for now. Today we digested both promoters and GFP , ligated them and transformed them using our electrocompetent TOP 10 E. coli.
-
| style="text-align: center;" |Primer RV
+
-
| style="text-align: center;" |2,5 ul
+
-
|-
+
=July 4=
-
| style="text-align: center;" |DMSO
+
All our transformations were unsuccessful. After running the digestions in a gel we realized that the digestion was not complete. We did some positive controls on all the enzymes to verify that they are working properly, but it all seems to be working fine.
-
| style="text-align: center;" |2,0 ul
+
-
|-
+
-
| style="text-align: center;" |Master Mix
+
-
| style="text-align: center;" |25 ul
+
-
|-
+
-
| style="text-align: center;" |dH2O
+
-
| style="text-align: center;" |16 ul
+
-
|-
+
-
|}
+
 +
=July 7=
-
We got a POSITIVE result on ''hoxN'', and POSITIVE with "R" at "low" temperatures of annealing. Here is our BEAUTIFULLL gel.
+
Today we are measuring fluorescence of the first part of the Interlab Study! We are also trying to begin joining the first two parts of our construct tetR inverter and mRFP. Here is some doodles we used to explain to the new iGemers how the process of digestion and ligation works and the logic behind it.
 +
=July 8=
-
{|align="center"
+
Primers have arrived! We are resuspending our primers and using them to make our first PCRs of the year. We are trying to get pqrr4 and LuxOD47E.  
-
|[[File:Gel1.jpg|300px|thumb|'''GoodNewsGEL''']]
+
-
|[[File:star.jpg|100px|thumb]]
+
-
|}
+
-
On 5,7,8,9 we can see "R" fragment of 359bp.
+
PCR products have just been confirmed with gel electrophoresis! Finally something is working!
-
On 12 we can see ''hoxN'' fragment of 837bp.
+
-
===='''11-Jul-2013'''====
+
=July 9=  
-
Today we did the PrcnA PCR and it didn't work out.
+
Ligation and transformation of the two remaining Interlab parts was a complete failure. We decided to purify the GFP fragment from the digestion and try using this to make the ligation.
 +
Now that we have the PCR products we are trying to make the following constructs: pqrr4-mRFP, pBAD-LuxOD47E, IL2-GFP, IL4-GFP.  
-
===='''15-Jul-2013'''====
+
=July 10=
-
We started ''hoxN'' PCR with the phusion primers, over the ''C. metallidurans'' genome, using 2-step PCR protocol.
+
Today we digested all the parts with the appropiate enzymes to make the constructs from yesterday. We ligated and trasnformed them. We also decided to start working on tetR inverte-mRFP and tetR inverter-amilCp.  
-
Results are negative.
+
=July 11=
-
===='''17-Jul-2013'''====
+
No colonies on any of the plates! :(.... We repeated all the transformations from yesterday. Hopefully, something will grow this time.
-
We repeated hoxN PCR with the fusion primers over the genome and PrcnA PCR with ''E.coli'' genome.
+
=Jul 14=
-
Nothing worked OUT :( !!!
+
We have colonies this time, but they are white. We were expecting red and blue colonies. After verifying the tetR inverter parts we got from the distribution kit, we found out that the size of the digested fragments do not correspond to the 902 bp or the 2070 bp of the inverter and pSB1C3. All three parts look the same. It seems we are not going to be able to use them.
-
We are going to try to do the PCR of hoxN with the phusion primers over the fragment already amplified and see, as fas as PrcnA goes...we will try a temperature ramp with a 3-step PCR protocol.
+
We are now looking into the possibility of using CI inverter instead of the tetR invertes. We are transforming the two CI invertes from the distrbution kit. Hopefully we'll better luck this time.  
-
End for today :(
+
-
===='''18-Jul-2013'''====
+
We have not been able to get the two remaining Interlab constructs.
-
Today we did ''E. coli'' chimiocompetent cells to mRFP transformation using TransforrmAid Bacterial Transformation Kit (Thermo Scientific). We did mRFP transformation from iGEM plate.
+
=July 15=
-
===='''19-Jul-2013'''====
+
We got colonies from CI inverter parts 18B and 18D on 2013 distribution kit! Anddd we got a colony of IL2-3 interlab part! We are making an ON of these parts so we can miniprep tomorrow!  
-
We have transformants!!!! Beautiful, aren´t they?
+
Also we transformed IL3-4, tetR inverter-amilCp,tetR-RFP on E.coli TOP 10.
-
[[File:IMG-20130909-00382.jpg|400px|thumb|center|Transformants]]
+
-
===='''24-Jul-2013'''====
+
=July 16=
-
<html>
+
-
<p align="justify">
+
-
Today we are going to do pRcnA PCR. Besides iGEM kit, we used Phusion High Fidelity PCR kit and tested  its two buffers: Phusion HF reaction Buffer and Phusion GC Reaction Buffer. We used a 2-step PCR protocol. Reagents and quantities of iGEM kit are the same we have been using until now, but reagents and quantities used for Phusion High Fidelity PCR kit are shown in the table below.
+
-
</p>
+
-
</html>
+
-
{| border="1" align="center"
+
-
|+'''PCR reagents and amounts for one 50 ul reaction'''
+
-
| style="text-align: center;" |Reagent
+
-
| style="text-align: center;" |Amount
+
-
|-  
+
We got colonies on tetR inverter-RFP and tetR inverter-amilCp transformation but unfortunately all our colonies are white :(…So…something is definitely not working with tetR inverter part.
-
| style="text-align: center;" |''Escherichia coli'' DNA
+
Things to do:
-
| style="text-align: center;" |4,0 ul
+
IL4-3 ligation
 +
Miniprep CI inverter 18B, RFP, AmilCp, pBAD-LuxOD-GFP
-
|-
+
Later that day..
-
| style="text-align: center;" |Primer FW
+
All the minipreps were good and arabinose 10% w/v solution was prepared to test our pBAD-LuxOD –GFP part. So in order to make the experiment on the fluorimeter tomorrow we left three ON cultures on the shaker.
-
| style="text-align: center;" |2,5 ul
+
-
|-
+
=July 17=
-
| style="text-align: center;" |Primer RV
+
-
| style="text-align: center;" |2,5 ul
+
Today we did digestion with SpeI and PstI to our tetR inverter part with buffer 2.1 because we need to confirm that we have the correct bps on the plasmid.
-
|-
+
We also did ligation of: tetR inverter-RFP,tetR inverter-amilCP and IL4-3 , after the ligation we transformed again, so we can have our colonies tomorrow!!! >D
-
| style="text-align: center;" |Buffer
+
Amilcp, RFP and GFP digestions were confirmed on gel and tetR inverter digestion is not right, again the digested fragments do not correspond to what they are supposed to. :( We are thinking there is no hope with this part.
-
| style="text-align: center;" |10 ul
+
Since we were depressed with tetR inverter, we tried the digestion (SpeI-PstI) with CI inverter (18B and 18D).
-
|-
+
-
| style="text-align: center;" |dNTPs
+
-
| style="text-align: center;" |1,0 ul
+
-
|-
+
-
| style="text-align: center;" |DMSO
+
-
| style="text-align: center;" |1,5 ul
+
-
|-
+
-
| style="text-align: center;" |Pol
+
-
| style="text-align: center;" |0,5 ul
+
-
|-
+
-
| style="text-align: center;" |dH2O
+
-
| style="text-align: center;" |28,3 ul
+
-
|-
+
-
|}
+
-
===='''25-Jul-2013'''====
+
-
We did ''hoxN'' PCR by using the same kits than yesterday and 2-steps protocol. However, results are negative :'(.
+
-
===='''26-Jul-2013'''====
+
=July 18=
-
<html>
+
-
<p align="justify">
+
-
Since we have been having troubles with PCRs, we decided to do genome extraction of our bacteria again. For this purpose we used Easy DNA Isolation Kit, Invitrogen. We followed manufacturer's instructions. We confirmed the extraction by gel electrophoresis. Genomes of ''C. metallidurans'' and new ''E.coli'' Extracted :D 
+
-
</html>
+
-
[[File:GelGenoma.jpg|400px|thumb|center|GenomeNewExtractionWithOUTRnase]]
+
-
<html>
+
-
</p>
+
-
</html>
+
-
*Remember you can review protocols in the section named [https://2013.igem.org/Team:Colombia_Uniandes/Protocols Protocols]
+
-
===='''29-Jul-2013'''====
+
We made the fluorescence experiment of our pBAD-LuxOD-GFP part and it didn’t work, also we did the digestion and iiiit didn’t work, so we have to make a new plan.
-
Today we did PCR of ''hoxN'' from the new ''C. metallidurans'' genome exracted and guess what? We got ''hoxN'' a beatiful band around 600 pb :D :D. Blue arrow shows HoxN and yellow arrow shows rcnR...FAIL
+
We have to do the following:
 +
Repeat pBAD-LuxOD-GFP ligation
 +
Transform pBAD-LuxOD-GFP ligation and tetR inverter AGAIN
 +
Miniprep pBAD-LuxOD-GFP and tetR inverter
 +
Digest pBAD-LuxOD-GFP , CI miniprep and tetR inverter with EcoRI and PstI
 +
Digest GFP with Xba-Pst
-
[[File:GelHoxN.jpg|400px|thumb|center|GelHoxN]]
+
=July 21=
 +
 +
There are some parts on the registry that could work for us:
 +
The first is RBS-tetR-ter-ter-Ptet-RBS-mRFP
 +
The second is Pcat-RBS-tetR-ter-ter-Ptet-RBS-mRFP
 +
We thought maybe we could order some primers and amplify only the RBS-tetR-ter-ter-Ptet-RBS-mRFP part of the second construct orrr even better the first one is just right for us in order to add the Pqrr4 promoter amplified by PCR previously!
 +
Let’s see how it goes! Today we transformed these parts and also if this doesn’t work we will try to make it all from scratch…transforming tetR alone, ter, Ptet and RBS.
-
===='''30-Jul-2013'''====
+
=July 22=
-
We did PCR of ''rcnR'' using the ''E. coli'' genome extracted last Friday and Phusion High Fidelity PCR kit. On the gel we got a diffuse band around 400 pb, so we could not conclude anything.
+
-
===='''31-Jul-2013'''====
+
tetR protein didn’t  work out. Nothing is working out :(
-
<html>
+
-
<p align="justify">
+
-
We did PCR of ''pRcnA'' and ''rcnR'' again using a annealing temperature gradient between 45°C and 60° and using the two buffers: Phusion HF reaction Buffer and Phusion GC Reaction Buffer. On the gel we saw a band that corresponded to ''pRcnA'' when the annealing temperature had been around 53°C and GC buffer had been used. On the other hand, in ''rcnR'' reactions we got a band when buffer HF had been used and when annealing temperature was around 58°C.
+
-
So...u CAN BARELY see them BUT THEY ARE THERE!
+
-
</html>
+
-
[[File:PrcnA.jpg|400px|thumb|center|Prcna,rcnR]]
+
-
<html>
+
-
</p>
+
-
</html>
+
-
===='''01-Aug-2013'''====
+
-
<html>
+
-
<p align="justify">
+
-
Today we did ''hoxN''-''rfp'' phusion. We used Phusion High Fidelity PCR kit, Phusion HF reaction Buffer and the amounts of other reagents described below. We got our first phusion!!! What a great day :)
+
-
</p>
+
-
</html>
+
-
{| border="1" align="center"
+
-
|+'''Phusion PCR'''
+
-
| style="text-align: center;" |Reagent
+
-
| style="text-align: center;" |Amount
+
-
|-
+
=July 25=
-
| style="text-align: center;" |''rfp''  DNA     
+
Digest: IL2, IL4 and IL2-3
-
| style="text-align: center;" |2,0 ul
+
Gel confirmation
-
|-  
+
Ligate : IL2-3, IL4-3
-
| style="text-align: center;" |''hoxN''  DNA
+
Transform : IL2-3, IL4-3
-
| style="text-align: center;" |2,0 ul
+
-
|-  
+
-
| style="text-align: center;" |Primer FW
+
-
| style="text-align: center;" |1,0 ul
+
-
|-
+
=July 28=
-
| style="text-align: center;" |Primer RV
+
-
| style="text-align: center;" |1,0 ul
+
-
|-
+
-
| style="text-align: center;" |Buffer
+
-
| style="text-align: center;" |4 ul
+
-
|-
+
-
| style="text-align: center;" |dNTPs
+
-
| style="text-align: center;" |0,4 ul
+
-
|-
+
-
| style="text-align: center;" |DMSO
+
-
| style="text-align: center;" |1,0 ul
+
-
|-
+
-
| style="text-align: center;" |Pol
+
-
| style="text-align: center;" |0,2 ul
+
-
|-
+
-
| style="text-align: center;" |dH2O
+
-
| style="text-align: center;" |8,4 ul
+
-
|-
+
-
|}
+
-
[[File:hoxNRFP.jpg|400px|thumb|center|hoxNRFP fusion!]]
+
RBS transformation and RBS-tetR-ter-ter-Ptet-RBS-mRFP in ampicilin BB didnt work out, there are some colonies in doubleTer petri dish and IL2-3 , IL4-3 have NOOO colonies at all.
-
===='''02-Aug-2013'''====
+
We repeated  IL2-3 , IL4-3 transformation in CM BB and RBS-tetR-ter-ter-Ptet-RBS-mRFP and RBS in AMP BB.
-
Another try of PrcnR-rcnR and then again...nothing.
+
Note: MilliQ Water and eppendorfs must be sterilized!!!
-
[[File:rcnR.jpg|400px|thumb|center|rcnR]]
+
=July 29=
 +
 +
We have colonies of:
 +
RBS-tetR-ter-ter-Ptet-RBS-mRFP
 +
doubleTer
 +
RBS
 +
Ptet
 +
IL2-3
-
===='''05-Aug-2013'''====
+
We repeated transformation of IL43 and left ON of the rest of the parts so we can miniprep tomorrow.
-
Since we obtained PrcnA and HoxN, we made a Prcna-HoxN fusion and we also amplified hoxN-RFP fusion. All was obtained and we can see that in our beautifull gel :)Go guys!! The fisrt column next to the DNA ladder is HoxN-RFP fusion on the first pink arrow, on the second pink arrow there is our fusion Prcna-HoxN.
+
=July 30=
-
[[File:Prcna_hoxN.jpg|400px|thumb|center|Prcna-HoxN and HoxN-RFP]]
+
Today we are going to make digestion of all the minipreps:
 +
RBS-tetR-ter-ter-Ptet-RBS-mRFP (EcoRI-PstI)
 +
doubleTer (EcoRI-Xba)
 +
RBS (EcoRI-PstI)
 +
Ptet (Spe-Pst)
 +
IL2-3 (Eco-PstI)
 +
IL4-3(Eco-PstI)
 +
tetR alone (Eco-Pst)
 +
And on our gel for today our lovely audience we haveeee:
 +
RBS-tetR-ter-ter-Ptet-RBS-mRFP (EcoRI-PstI): Many many bands
 +
doubleTer (EcoRI-Xba): CONFIRMED
 +
RBS (EcoRI-PstI): CONFIRMED
 +
Ptet (Spe-Pst): CONFIRMED
 +
IL2-3 (Eco-Spe): CONFIRMED
 +
IL4-3(Eco-PstI): CONFIRMED
 +
tetR alone: CONFIRMED
 +
We made Ptet-RFP and Ptet-amilCP ligation :)
 +
=July 31=
-
===='''07-Aug-2013'''====
+
We made ON of IL43, IL23 and pBAD-LuxOD-GFP for fluorimeter
 +
Ligate RBS-tetR alone and tetR alone-doubleTer
 +
Transform Ptet-RFP and Ptet-amilCp
-
Today we decided to digest our parts with EcoRI in order to insert our parts in the backbone. Thats why first we did the digestion with EcoRI on the Backbone and also the same process with the parts. We digested for 2 hours and then desactivated the ennzymes at 80°C for 20 min. For protocol see: https://www.neb.com/products/r0101-ecori#tabselect2
+
=August 4=
 +
 +
Miniprep Ptet-RFP, RFP, tetR-dter,amilcp,tet alone
 +
Digest RBS,RFP,amilcp,tet-dter,tet alone
 +
Ligate RBS-tetR,RBS-tet-dter
 +
Transform
-
We then used antarctic phosphatase for the backbone for 1h. For protocol see: https://www.neb.com/protocols/1/01/01/vector-dephosphorylation-protocol
+
=August 6=
 +
Up until now we have IL1, IL23 and IL34 parts for interlab and we are waiting for our turn in the fluorimeter. Also we have tetR, tetR-dter,Ptet-RFP and Ptet-amilcp.
-
Finally we let the ligation all night at room temperature.
+
=August 8=
 +
 +
Digestions…digestions…sad faces…sad faces…we want to go home….we want to go home..
-
===='''09-Aug-2013'''====
+
=August 11=
-
Today we desactivated the ligase at 80°C for 15 min and started THE TRANSFORMATION!
+
We are trying to do the same thing all over and over again and we are going crazy. For real.
-
We transformated our cells and let them in 37°C. Lets see tomorrow :)
+
=August 12=
 +
We made ON of our transformant colonies in RBS-tetR and tetR-dt and also we did our fluorescence experiment today on IL23, pBAD-LuxOD-GFP and IL43. Results will be showed later alligatorrsss!
-
===='''10-Aug-2013'''====
+
=August 13=
-
They were NO colonies At ALL! not even halfffffff, not even a tiny oneee!!
+
-
We are repeating process on monday.
+
NOT GOOD NEWS....Our negative control (DH5alfa) gave the same results as our beloved parts on IL23, pBAD-LuxOD-GFP and IL43, soooo we will have to go over again.
 +
We did miniprep today of IL23,IL43, pSB1A3+RFP and tetR-dter.
 +
Also we digested the linearized plasmid of Amp and also tetR with Eco-Pst
-
===='''13-Aug-2013'''====
+
Digestions:
-
Today we did electrocompetent cells with E.coli DH10B and we tranformed and well...hopefully we will see something tomorrow!
+
IL23 (Eco-Pst)
 +
IL43 (Eco-Pst)
 +
tetR (Eco-Spe)
 +
pSB1A3+RFP (Eco-Pst)
 +
pSB1A3 ln (Eco-Pst)
-
Hopes up!
+
ligations:
 +
tetR-dter in pSB1A3+RFP
 +
tetR-dter in pSB1A3 ln
-
===='''14-Aug-2013'''====
+
Transform:
 +
tetR-dter in pSB1A3+RFP
 +
tetR-dter in pSB1A3 ln
 +
RBS-tetR in kan plasmid
 +
pSB1C3+RFP
 +
pSB1K3+RFP
-
We have some colonies! but only on HoxN-RFP fusion! We are going to leave those on ON and we are transforming Prcna-HoxN again.
+
We are going to use the pSB1C3+RFP and pSB1K3+RFP plasmids for convinience, so we can see that our part inserted the plasmid by losing its color and appearing white on the petri dish.
-
===='''15-Aug-2013'''====
+
IL23 and IL43 digestions did not work. All of the others did.
-
Transformation not succesfull, do it one more time!
+
=August 14=
-
===='''16-Aug-2013'''====
+
We did miniprep of pSB1C3+RFP and pSB1K3+RFP.
-
Transformation not succesfull, do it one more time!
+
Our minipreps are not going good,its weird.. the concentrations are very very low and we dont know if is the lysis solution. We are going to try working with double of the amount required of lysis solution and also we are going to warm up the water in order to see if the concentration gets higher.
-
===='''19-Aug-2013'''====
+
=August 15=
-
Transformation not succesfull, do it one more time!
+
Today, transmitting  from one computer to another!
 +
We did miniprep of pSB1K3+RFP, pSB1K3+RFP and tetR-dTer...but once again minipreps are not working properly.
-
===='''20-Aug-2013'''====
+
We thought today in a new possible construct, and this time the revoolutionary part is a feedback! it would be like this..
-
Tranformation succesfull!!!! :) Growing on ON!
+
[[File:Screen_Shot_2014-10-17_at_10.55.30_PM.png|center|600px]]
-
===='''21-Aug-2013'''====
+
In this way we can se color much more easier and faster because the reporter is under 2 promoters that activate on the same situation.
-
Miniprep DAY! :)
+
For this we transformed--> Psid (BBa_I746364) Inducible promoter, activated by psp3 protein
 +
                          RBS+Psp3 (BBa_I746351) Activator of Psid promoter
-
Parts Ready!
+
=In our final month=
-
===='''23-Aug-2013'''====
+
We amplified by PCR RBS-tetR-dter-Ptet-RFP out of the registry part with pCAT.
 +
We amplified Pqrr4 for ligation with the final construct, we still havent succeded by ligating this promoter to the construct.
 +
We obtained pBAD-LUXOD-GFP and made the fluorescence measurements.
 +
We obtained the Psid-Ptet-rbs-RFP-psp3 and we are now workig on a experiment to prove that the system work transforming with tetR protein in a different plasmid under a constitutive promoter and Psid-Ptet-rbs-RFP-psp3 in the same E.coli, by growing this bacteria under different tetracicline concentrations a gradient of color should be obtained.
 +
We obtained rbs-tetR-dter-Pter-RFP
-
We are sending Prcna-HoxN and HoxN-RFP :)
+
Also we are still trying to ligate Pqrr4 promoter to the construct Psid-Ptet-rbs-RFP-psp3 for our final results.
-
We are still trying to get Prcnr-rcnR so lets go!
+
-
 
+
-
===='''24-Aug-2013'''====
+
-
 
+
-
We tried PCR Prcnr-rcnR and IS NOT THEREEEEEEEEEEEE!
+
-
<html>
+
-
</p>
+
-
    </div>
+
-
  </div>
+
-
</div>
+
-
 
+
-
 
+
-
 
+
-
</div>
+
-
</div>
+
-
</html>
+

Latest revision as of 03:59, 18 October 2014

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Journal



Fotojour.jpg

Contents

June 24

Today was the first day at the lab!! Finally! After finding out the parts we ordered from the registry had been sitting in a locker for the last two weeks and managing to salvage them we re finally up and running. First thing first, today we are making electrocompetent cells and transforming our first parts. We are using TOP 10 strain to make our transformations. We are transforming mRFP (BBa_K518012), pBAD (BBa_K206000), tetR inverter (BBa_Q04400), amilCp (BBa_K592025), pBAD-CqsA (BBa_K581011), pTet-LuxOD47E (BBa_K218017), pqrr4-GFP (BBa_K581009) and LuxU-CqsS-LuxO (BBa_K581010). We are starting with the Interlab project, so we are transforming those parts as well. For convenience, we have decided to name the Interlab parts IL1 (BBa_I20260, promoter+GFP in 1K3), IL2 (BBa_J23101, promoter), IL3 (E0240, GFP) and IL4 (BBa_J23115, promoter).

We just realized we are all out of our kanamycin stock. We need to prepare some more tomorrow.

June 25

We have transformants! Well, almost. Nothing grew on the IL4 plate :(. But nothing to worry about, we are just repeating the transformations. We made passes to liquid media of single colonies of all the other parts and we are hoping to do miniprep of all them tomorrow.

We also cryopreserved all the bacteria we got from the registry. Just made a fresh new Kanamycin stock, so we can now transform tetR inverter.

June 26

Our first minipreps were unsuccessful and nothing grew on the tetR inverter plate.

We just realized the tetR inverter part has two other twins in the registry. We are transforming those as well and hope one of them works.

June 27

Today we prepared homemade lysis buffer for our minipreps, but apparently it wasn't very effective. We could not see any DNA after running our miniprerps in a gel.

July 1

After a long weekend, we are back in business. We have colonies in all the plates with the trnasformations of the tetR inverter! We made passes of all three parts to liquid LB for miniprep tomorrow.

July 2

We repeated the miniprep of all the parts and finally got it right this time! All molecular weights correspond to what we expected!

July 3

Now that we have all the parts, we can start building our construct! We are starting with the Interlab parts for now. Today we digested both promoters and GFP , ligated them and transformed them using our electrocompetent TOP 10 E. coli.

July 4

All our transformations were unsuccessful. After running the digestions in a gel we realized that the digestion was not complete. We did some positive controls on all the enzymes to verify that they are working properly, but it all seems to be working fine.

July 7

Today we are measuring fluorescence of the first part of the Interlab Study! We are also trying to begin joining the first two parts of our construct tetR inverter and mRFP. Here is some doodles we used to explain to the new iGemers how the process of digestion and ligation works and the logic behind it.

July 8

Primers have arrived! We are resuspending our primers and using them to make our first PCRs of the year. We are trying to get pqrr4 and LuxOD47E.

PCR products have just been confirmed with gel electrophoresis! Finally something is working!

July 9

Ligation and transformation of the two remaining Interlab parts was a complete failure. We decided to purify the GFP fragment from the digestion and try using this to make the ligation. Now that we have the PCR products we are trying to make the following constructs: pqrr4-mRFP, pBAD-LuxOD47E, IL2-GFP, IL4-GFP.

July 10

Today we digested all the parts with the appropiate enzymes to make the constructs from yesterday. We ligated and trasnformed them. We also decided to start working on tetR inverte-mRFP and tetR inverter-amilCp.

July 11

No colonies on any of the plates! :(.... We repeated all the transformations from yesterday. Hopefully, something will grow this time.

Jul 14

We have colonies this time, but they are white. We were expecting red and blue colonies. After verifying the tetR inverter parts we got from the distribution kit, we found out that the size of the digested fragments do not correspond to the 902 bp or the 2070 bp of the inverter and pSB1C3. All three parts look the same. It seems we are not going to be able to use them.

We are now looking into the possibility of using CI inverter instead of the tetR invertes. We are transforming the two CI invertes from the distrbution kit. Hopefully we'll better luck this time.

We have not been able to get the two remaining Interlab constructs.

July 15

We got colonies from CI inverter parts 18B and 18D on 2013 distribution kit! Anddd we got a colony of IL2-3 interlab part! We are making an ON of these parts so we can miniprep tomorrow! Also we transformed IL3-4, tetR inverter-amilCp,tetR-RFP on E.coli TOP 10.

July 16

We got colonies on tetR inverter-RFP and tetR inverter-amilCp transformation but unfortunately all our colonies are white :(…So…something is definitely not working with tetR inverter part. Things to do: IL4-3 ligation Miniprep CI inverter 18B, RFP, AmilCp, pBAD-LuxOD-GFP

Later that day.. All the minipreps were good and arabinose 10% w/v solution was prepared to test our pBAD-LuxOD –GFP part. So in order to make the experiment on the fluorimeter tomorrow we left three ON cultures on the shaker.

July 17

Today we did digestion with SpeI and PstI to our tetR inverter part with buffer 2.1 because we need to confirm that we have the correct bps on the plasmid. We also did ligation of: tetR inverter-RFP,tetR inverter-amilCP and IL4-3 , after the ligation we transformed again, so we can have our colonies tomorrow!!! >D Amilcp, RFP and GFP digestions were confirmed on gel and tetR inverter digestion is not right, again the digested fragments do not correspond to what they are supposed to. :( We are thinking there is no hope with this part. Since we were depressed with tetR inverter, we tried the digestion (SpeI-PstI) with CI inverter (18B and 18D).

July 18

We made the fluorescence experiment of our pBAD-LuxOD-GFP part and it didn’t work, also we did the digestion and iiiit didn’t work, so we have to make a new plan. We have to do the following: Repeat pBAD-LuxOD-GFP ligation Transform pBAD-LuxOD-GFP ligation and tetR inverter AGAIN Miniprep pBAD-LuxOD-GFP and tetR inverter Digest pBAD-LuxOD-GFP , CI miniprep and tetR inverter with EcoRI and PstI Digest GFP with Xba-Pst

July 21

There are some parts on the registry that could work for us: The first is RBS-tetR-ter-ter-Ptet-RBS-mRFP The second is Pcat-RBS-tetR-ter-ter-Ptet-RBS-mRFP We thought maybe we could order some primers and amplify only the RBS-tetR-ter-ter-Ptet-RBS-mRFP part of the second construct orrr even better the first one is just right for us in order to add the Pqrr4 promoter amplified by PCR previously! Let’s see how it goes! Today we transformed these parts and also if this doesn’t work we will try to make it all from scratch…transforming tetR alone, ter, Ptet and RBS.

July 22

tetR protein didn’t work out. Nothing is working out :(

July 25

Digest: IL2, IL4 and IL2-3 Gel confirmation Ligate : IL2-3, IL4-3 Transform : IL2-3, IL4-3

July 28

RBS transformation and RBS-tetR-ter-ter-Ptet-RBS-mRFP in ampicilin BB didnt work out, there are some colonies in doubleTer petri dish and IL2-3 , IL4-3 have NOOO colonies at all.

We repeated IL2-3 , IL4-3 transformation in CM BB and RBS-tetR-ter-ter-Ptet-RBS-mRFP and RBS in AMP BB. Note: MilliQ Water and eppendorfs must be sterilized!!!

July 29

We have colonies of: RBS-tetR-ter-ter-Ptet-RBS-mRFP doubleTer RBS Ptet IL2-3

We repeated transformation of IL43 and left ON of the rest of the parts so we can miniprep tomorrow.

July 30

Today we are going to make digestion of all the minipreps: RBS-tetR-ter-ter-Ptet-RBS-mRFP (EcoRI-PstI) doubleTer (EcoRI-Xba) RBS (EcoRI-PstI) Ptet (Spe-Pst) IL2-3 (Eco-PstI) IL4-3(Eco-PstI) tetR alone (Eco-Pst) And on our gel for today our lovely audience we haveeee: RBS-tetR-ter-ter-Ptet-RBS-mRFP (EcoRI-PstI): Many many bands doubleTer (EcoRI-Xba): CONFIRMED RBS (EcoRI-PstI): CONFIRMED Ptet (Spe-Pst): CONFIRMED IL2-3 (Eco-Spe): CONFIRMED IL4-3(Eco-PstI): CONFIRMED tetR alone: CONFIRMED We made Ptet-RFP and Ptet-amilCP ligation :)

July 31

We made ON of IL43, IL23 and pBAD-LuxOD-GFP for fluorimeter Ligate RBS-tetR alone and tetR alone-doubleTer Transform Ptet-RFP and Ptet-amilCp

August 4

Miniprep Ptet-RFP, RFP, tetR-dter,amilcp,tet alone Digest RBS,RFP,amilcp,tet-dter,tet alone Ligate RBS-tetR,RBS-tet-dter Transform

August 6

Up until now we have IL1, IL23 and IL34 parts for interlab and we are waiting for our turn in the fluorimeter. Also we have tetR, tetR-dter,Ptet-RFP and Ptet-amilcp.

August 8

Digestions…digestions…sad faces…sad faces…we want to go home….we want to go home..

August 11

We are trying to do the same thing all over and over again and we are going crazy. For real.

August 12

We made ON of our transformant colonies in RBS-tetR and tetR-dt and also we did our fluorescence experiment today on IL23, pBAD-LuxOD-GFP and IL43. Results will be showed later alligatorrsss!

August 13

NOT GOOD NEWS....Our negative control (DH5alfa) gave the same results as our beloved parts on IL23, pBAD-LuxOD-GFP and IL43, soooo we will have to go over again. We did miniprep today of IL23,IL43, pSB1A3+RFP and tetR-dter. Also we digested the linearized plasmid of Amp and also tetR with Eco-Pst

Digestions: IL23 (Eco-Pst) IL43 (Eco-Pst) tetR (Eco-Spe) pSB1A3+RFP (Eco-Pst) pSB1A3 ln (Eco-Pst)

ligations: tetR-dter in pSB1A3+RFP tetR-dter in pSB1A3 ln

Transform: tetR-dter in pSB1A3+RFP tetR-dter in pSB1A3 ln RBS-tetR in kan plasmid pSB1C3+RFP pSB1K3+RFP

We are going to use the pSB1C3+RFP and pSB1K3+RFP plasmids for convinience, so we can see that our part inserted the plasmid by losing its color and appearing white on the petri dish.

IL23 and IL43 digestions did not work. All of the others did.

August 14

We did miniprep of pSB1C3+RFP and pSB1K3+RFP.

Our minipreps are not going good,its weird.. the concentrations are very very low and we dont know if is the lysis solution. We are going to try working with double of the amount required of lysis solution and also we are going to warm up the water in order to see if the concentration gets higher.

August 15

Today, transmitting from one computer to another!

We did miniprep of pSB1K3+RFP, pSB1K3+RFP and tetR-dTer...but once again minipreps are not working properly.

We thought today in a new possible construct, and this time the revoolutionary part is a feedback! it would be like this..

Screen Shot 2014-10-17 at 10.55.30 PM.png

In this way we can se color much more easier and faster because the reporter is under 2 promoters that activate on the same situation.

For this we transformed--> Psid (BBa_I746364) Inducible promoter, activated by psp3 protein 

                          RBS+Psp3 (BBa_I746351) Activator of Psid promoter

In our final month

We amplified by PCR RBS-tetR-dter-Ptet-RFP out of the registry part with pCAT. We amplified Pqrr4 for ligation with the final construct, we still havent succeded by ligating this promoter to the construct. We obtained pBAD-LUXOD-GFP and made the fluorescence measurements. We obtained the Psid-Ptet-rbs-RFP-psp3 and we are now workig on a experiment to prove that the system work transforming with tetR protein in a different plasmid under a constitutive promoter and Psid-Ptet-rbs-RFP-psp3 in the same E.coli, by growing this bacteria under different tetracicline concentrations a gradient of color should be obtained. We obtained rbs-tetR-dter-Pter-RFP

Also we are still trying to ligate Pqrr4 promoter to the construct Psid-Ptet-rbs-RFP-psp3 for our final results.

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