Team:Colombia/Journal

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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">Mes # 1</a></li>
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<br><br>
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    <li><a href="#tab2" data-toggle="tab">Mes # 2</a></li>
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</html>
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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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=='''CONTENIDO'''==
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<p align="justify">
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Lalalala.
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</p>
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===='''CONTENIDO 2'''====
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[[File:Fotojour.jpg|center|600px]]
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<p align="justify">
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Lalalalla
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</p>
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<ol>
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<li>ej1</li>
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<li>ej2</li>
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<li>ej3</li>
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</ol>
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=June 24=
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==== '''15th June 2013''' ====
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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).
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We picked the transformant colonies.
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We just realized we are all out of our kanamycin stock. We need to prepare some more tomorrow.
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===='''18th June 2013'''====
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=June 25=
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<p>We performed miniprep procedures with the GenElute HP Plasmid Miniprep kit.</p>
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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.
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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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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.  
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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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=June 26=
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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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Our first minipreps were unsuccessful and nothing grew on the tetR inverter plate.  
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===='''June 27, 2013'''====
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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.  
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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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=June 27=
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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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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.
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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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=July 1=
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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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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.  
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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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=July 2=
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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 repeated the miniprep of all the parts and finally got it right this time! All molecular weights correspond to what we expected!
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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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=July 3=
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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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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.
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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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=July 4=
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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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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.
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===='''July 18th, 2013'''====
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=July 7=
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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 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.
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Today we amplified F2 that we obtained yesterday.
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===='''July 22nd, 2013'''====
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=July 8=
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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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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.  
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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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PCR products have just been confirmed with gel electrophoresis! Finally something is working!  
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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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=July 9=  
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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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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.
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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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Now that we have the PCR products we are trying to make the following constructs: pqrr4-mRFP, pBAD-LuxOD47E, IL2-GFP, IL4-GFP.  
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===='''August 17th,2013'''====
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=July 10=
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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 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.  
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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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=July 11=
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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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No colonies on any of the plates! :(.... We repeated all the transformations from yesterday. Hopefully, something will grow this time.
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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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=Jul 14=
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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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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.
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After transforming them we got these minipreps
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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.  
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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 have not been able to get the two remaining Interlab constructs.
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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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=July 15=
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===='''September 3rd, 2013'''====
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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!  
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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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Also we transformed IL3-4, tetR inverter-amilCp,tetR-RFP on E.coli TOP 10.
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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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=July 16=
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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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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.
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Everything is ready for the validation experiments. We're both nervous and excited!
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Things to do:
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IL4-3 ligation
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Miniprep CI inverter 18B, RFP, AmilCp, pBAD-LuxOD-GFP
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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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Later that day..
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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.  
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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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=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.
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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
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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.
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Since we were depressed with tetR inverter, we tried the digestion (SpeI-PstI) with CI inverter (18B and 18D).
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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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=July 18=
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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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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.
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We have to do the following:
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Repeat pBAD-LuxOD-GFP ligation
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Transform pBAD-LuxOD-GFP ligation and tetR inverter AGAIN
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Miniprep pBAD-LuxOD-GFP and tetR inverter
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Digest pBAD-LuxOD-GFP , CI miniprep and tetR inverter with EcoRI and PstI
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Digest GFP with Xba-Pst
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=July 21=
 +
 +
There are some parts on the registry that could work for us:
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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.
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<html></p>
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=July 22=
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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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=='''COTENIDO OTRO MES'''==
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tetR protein didn’t  work out. Nothing is working out :(
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<html>
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<p align="justify">
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Lalalala
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</p>
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</html>
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<html>
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=July 25=
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</p>
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Digest: IL2, IL4 and IL2-3
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    </div>
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Gel confirmation
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  </div>
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Ligate : IL2-3, IL4-3
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</div>
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Transform : IL2-3, IL4-3
 +
=July 28=
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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.
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</div>
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We repeated  IL2-3 , IL4-3 transformation in CM BB and RBS-tetR-ter-ter-Ptet-RBS-mRFP and RBS in AMP BB.
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</div>
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Note: MilliQ Water and eppendorfs must be sterilized!!!
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</html>
+
 
 +
=July 29=
 +
 +
We have colonies of:
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RBS-tetR-ter-ter-Ptet-RBS-mRFP
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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)
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Ptet (Spe-Pst)
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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
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Digest RBS,RFP,amilcp,tet-dter,tet alone
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Ligate RBS-tetR,RBS-tet-dter
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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..
 +
 
 +
[[File:Screen_Shot_2014-10-17_at_10.55.30_PM.png|center|600px]]
 +
 
 +
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.

Latest revision as of 03:59, 18 October 2014

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Journal



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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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