Team:KU Leuven/Journal/MeS/wetlab

From 2013.igem.org

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   <h3 class="bg-red">Methylsalicylate biobrick</h3>
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   <p align="justify">This is the journal for the MeS producing biobrick, a part of the glucose model. For more information about this biobrick we refer you to the glucose model page.</p>
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   <p align="justify">This is the journal for the MeS producing biobricks, part of the glucose model. For more information about these bricks and the model we refer you to the <a href="https://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel">glucose model page</a>.</p>
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Revision as of 14:37, 4 October 2013

iGem

Secret garden

Congratulations! You've found our secret garden! Follow the instructions below and win a great prize at the World jamboree!


  • A video shows that two of our team members are having great fun at our favourite company. Do you know the name of the second member that appears in the video?
  • For one of our models we had to do very extensive computations. To prevent our own computers from overheating and to keep the temperature in our iGEM room at a normal level, we used a supercomputer. Which centre maintains this supercomputer? (Dutch abbreviation)
  • We organised a symposium with a debate, some seminars and 2 iGEM project presentations. An iGEM team came all the way from the Netherlands to present their project. What is the name of their city?

Now put all of these in this URL:https://2013.igem.org/Team:KU_Leuven/(firstname)(abbreviation)(city), (loose the brackets and put everything in lowercase) and follow the very last instruction to get your special jamboree prize!

tree ladybugcartoon

This is the journal for the MeS producing biobricks, part of the glucose model. For more information about these bricks and the model we refer you to the glucose model page.

  • Week 3: Something smells!

    This week we transformed DH5α cells with the methylsalicylate producing brick from MIT 2006 (Bba_J45700). Since this brick has got two antibiotic resistance genes (kanamycin and ampicillin), we plated them out on kanamycin plates and ampicillin plates.
    Using the cells containing Bba_J45700, some of our team members did a smell test, to see whether the production of methylsalicylate was significant, since methylsalicylate has a wintergreen scent. Plate C was the one containing these cells. These are the results, “-“ meaning no difference in scent compared to the control, “+” meaning a difference in scent compared to the control. As can be seen, plate C is chosen only a few times. The brick might need some improvement, for example by using stronger promoters and ribosome binding sites. So we will reconstruct Bba_J45700 by taking out the useful pieces and putting them back together with other parts.

    Nameplate Aplate Bplate C
    Ingmar+--
    Su+--
    Sander+--
    Bert---
    Frederik+++
    Saar--+
    Robbert+--
    Sabine+-+
    Flore++-

    In order to get pchBA and bsmt1 out of the original brick, a PCR on pchBA and bsmt1 is done. The fragments are cut with EcoRI and PstI, ligated in pSB1C3 and transformed.

  • Week 4: aroG, bsmt1 and pchBA, the holy trinity.

    Last week’s transformation failed, so we did PCR purification to make sure that all the small fragments were gone. The transformation was repeated.
    To improve the production of methylsalicylate, we wanted to improve the production of chorismate, a precursor of methylsalicylate. The product of aroG is an important enzyme in the production of chorismate. We first wanted to isolate this aroG, and then mutate it to make it insensitive to negative feedback from phenylalanine.
    We did a colony PCR on 4 random DH5α, to amplify aroG.
    Fragments were all around 1000 bp, as expected.
    Restriction with EcoRI and PstI, ligation and transformation.


    ebf gel1

    Our transformations for aroG, bsmt1 and pchBA were all succesful. To check whether the genes were actually inserted into the plasmids, we did a colony PCR on all three (in five-fold, five colonies of each gene).
    Unfortunately, only the aroG band had the right length. For the other two genes, only very small fragments were visible. We inoculated the aroG containing cells.
    For the mutagenesis of aroG, we performed a mutagenesis PCR with primers with a point mutation in them. After this, we did a KLD treatment (kinase, ligase, DpnI) + transformation.

  • Week 5: Everything fails!

    Mutagenesis didn’t work since there was no PCR product to be seen on a gel. We performed the mutagenesis with a high fidelity polymerase in a gradient PCR with different annealing temperatures (53-55-58-61-64°C) in parallel.


    ebf gel2

    On a gel, we put:

    • lane 2: the original plasmid containing aroG
    • lane 3: the original plasmid containing aroG cut with EcoRI to make it linear
    • lanes 4-8: the PCR products of the gradient PCR, one for each annealing temperature

    After visualisation, there were PCR products visible with lengths corresponding to the length of the cut plasmid, as expected.
    After ligation and transformation, there were no colonies to be seen, so we repeated this with an extra DpnI treatment to make sure that all the original plasmid was cut into smaller fragments that wouldn’t be able to be transformed. Unfortunately, also this transformation was unsuccesful.

  • Week 6: Mutations

    We decided to do something different to try and mutate aroG, by making us of a megaprimer. This megaprimer was made with a regular primer, starting from the biobrick suffix, and a primer containing a point mutation.
    In order to get rid of the primers that were used in the PCR reaction of this megaprimer, we put the product on a gel containing crystal violet. The purified megaprimer was used in a PCR reaction with the aroG containing plasmid, in order to mutate it.

    35µlH2O
    10µlbuffer
    0.5µldNTP
    2µlmegaprimer
    1.5µlaroG plasmid
    1µlPhire polymerase
    For the mutagenesis of aroG, we ran a PCR reaction without polymerase, to have a comparison on the gel.
    No bands were visible for either the positive and the negative mutagenesis PCR reaction. However, it is possible that a cluster of annealed plasmids didn't move into the gel, since there is a high signal in the well. Luckily, Misha told us, E.coli is capable of untangling this, so a transformation might still work.
    We also put the methylsalicylate producing brick into a new backbone, pSB1C3.
    In order to reconstruct the methylsalicylate brick for higher production, we also transformed the bricks 13B5 and 1M3, respectively pTetR+RBS and the lacI coding sequence on ampicillin and chloramphenicol plates, respectively.

  • Week 7: Megaprimer

    We started the construction of new biobricks:

    • pTetR + strong RBS
    • lacI + strong RBS
    • strong RBS + MIT 2006 brick

    The methylsalicylate producing brick (MIT 2006) in the pSB1C3 backbone was cut with EcoRI and PstI and put on gel. (Picture below, second lane, two bands: approximately 2000 and 3000 bp) We will now send this brick to be sequenced, so we will get a confirmation that this is indeed an improved brick.
    For the mutation of aroG, we repeated the mutagenesis PCR with the megaprimers,one reaction with polymerase (+) and one without (-).
    One part of this was put on gel (picture below).
    • lane 3: (-)
    • lane 4: (+)
    • lane 5: megaprimer
    • lane 6: aroG containing plasmid
    • gel

      The rest was DpnI treated, and dialysed to remove salts, by adding it to a 2% agarose + water gel.
      After 30 minutes the contents of the well were pipetted out and electroporated.
      Furthermore we performed a Phire II PCR reaction on our methyl salicylate brick, to obtain good PCR products of the bsmt1 and pchBA genes seperately (we need to get rid of the lacI operon in between, since it would interfere with the rest of the model).
      Our reaction composition was as follows:

      10 µl5X Phire Reaction Buffer
      1,5 µlForward primer
      1,5 µlReverse primer
      1 µldNTPs (10mM)
      1 µlTemplate DNA
      1 µlPhire II Polymerase
      34 µlMilli-Q water

      The PCR program we used was:

      98°C30s
      98°C5s |
      X °C5s | x35
      72°CYs |
      72°C60s
      4°Chold
      • X = 70°C for bsmt1, 73°C for pchBA
      • Y = 20s for bsmt1, 50s for pchBA
  • Week 8: Cut & Paste

    This week we performed different digestions and ligations to obtain several constructs for our final plasmids.:

    1. aroGM with E and S
    2. TT with E and X
    3. pTetR + RBS with E and S
    4. lacI + RBS with E and X
    5. pchBA PCR product with E and P
    6. pchBA PCR product with E and S
    7. bsmt1 PCR product with E and P
    8. bsmt1 PCR product with E and S
    After digestion we performed 6 different ligations:
    1. aroGM (E+S) with TT (E+X)
    2. pTetR+RBS (E+S) with lacI+RBS (E+X) in a
    3. pchBA (E+P) with Ampicillin backbone (E+P)
    4. bsmt1 (E+P) with Ampicillin backbone (E+P)
    5. bsmt1 (E+S) with TT (E+X)
    6. pchBA (E+S) with TT (E+X)
    7. After ligation, we performed chemical transformation of these ligation products in Inoue treated competent cells (TOP10). For 1, 5 and 6 we use plates containing chloramphenicol, for 2, 3 and 4 we use plates containing ampicillin.

  • Week 9: Last resort

    Unfortunately, after extracting these plasmids and cutting them with EcoRI and PstI, we didn’t see the expected bands. We repeated the digestions and ligations of the previous constructs, but without succes.
    This week, we started working on designing the gBlocks we were going to order. For the construct with pTetR-RBS-lacI-RBS-aroGM-TT we used 4 gBlocks, for the improvement of Bba-J45700, we used just one that contained a ptetR sequence in stead of a lac-promoter that would be affected by the lac repressor. The rest of the brick (the two parts to the side of this gBlock) was made with PCR of the original brick.