Team:Yale/Notebook

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    • Lab Notebook

    Outline of Our Project

    Outline of Our Project Goal: Create a recombinant protein using a dopamine surface anchor and an anti-fouling head-domain that can be expressed in recoded E. Coli.


    • Create an expression system
      • Creating theT7 Riboregulated System
        • First, create a construct that places the gene for T7 RNA polymerase in the backbone of a plasmid with a cis-repressing and trans-activating RNA.
          • Obtain T7 RNA Pol: from team strain #5.
          • Obtain pZE21 backbone: from Ryan
          • Design primers for PCR and Gibson Assembly (191, 192, 193, 194, 195)
          • PCR amplify T7 RNA Pol an pZE21 using primers.
          • DPN1 Digest template DNA.
          • Run fragments on a gel green Gel
          • Gel Purification of fragments.
          • Gibson Assembly of fragments into plasmid construct.
          • Transform plasmid construct into K12 derivative, ECNR2. Plate on Kan Plates.
          • Pick colonies from Kan Plates, inoculate liquid culture.
          • From liquid culture make a frozen stock.
          • Screen colonies for presence of plasmid of interest
            • First find which colonies have T7 and pZE21.
            • Use sequencing primers to amplify sequence of interest. Sequence for verification.
          • Integrate into genome of ECNR2 or BL21.
      • Create Promoter for T7 Riboregulated System
        • Create a construct that installs sfGFP behind promoter for T7.
          • Obtained T7 promoter from registry and Life Technologies.
          • obtained sfGFP gene from laboratory stock.
          • Approach 1: T7 Overhangs Approach
            • amplify standard pZE21::sfGFP plasmid with primers designed by Stephanie that have T7 as overhangs. Amplification of this plasmid will situate the T7 promoter in front of sfGFP.
            • DPN1 digest.
            • Gibson Assemble to circularize into plasmid.
            • Drop Dialysis to minimize salt.
            • transform into ECNR2/BL21
            • Screening for Plasmid
              • Insert plasmid into BL21 DE3 and screen for expression of sfGFP. BL21 DE3 is a strain that contains the gene for T7 RNA Polymerase.
          • Approach 2: Insert sfGFP into T7 containing plasmid.
            • Using psB1A2 with T7 promoter from registry. Transform bacteria to make stock of pSB1AC2.
            • Using primers designed by Ariel amplify pSB1AC2 to have overhangs with sfGFP.
            • Amplify sfGFP from pZE21::sfGFP with primers designed by Ariel to have overhangs with pSB1A2 with T7 Promoter.
            • DPN1 Digest
            • Gibson Assemble.
            • Drop Dialysis.
            • Transform into BL21/576
            • Screen for ampicillin/carbenicillin resistance.
            • Insert plasmid into BL21 DE3 and screen for sfGFP expression.
    • Create constructs
    • Express Constructs in rE.Coli
    • Test Constructs using Antimicrobial Assays
    • Test Constructs using Adhesion Assays.

    TroubleShooting (T7)

    Okay, so we’re currently having some issues with the transformations, so let’s take a step back and look at what can go wrong, and how we can fix them: 


    Template generation: For T7 RNA pol+pZE21 with the cr/ta system, the hairpins on the primers limit the efficacy of PCR and Gibson assembly.

    Solutions: First, we should screen the plasmids we have to check and see if they do in fact contain T7. Ariel has designed primers for the test already, and if necessary we can use the pZE21 with CAT as a control, since the band would be a significantly shorter length. 
         We can also try a different method of assembly: restriction enzyme digest (may be difficult because there are none that anneal EXACTLY to the spots we want, some are a few base pairs off), or deactivating CAT’s start codon and placing T7 a little ways downstream of the crRNA sequence, INSIDE CAT (which, without the start codon, would not be translated).

    Transformation: We need to drop dialyze longer. Our bacteria doesn’t seem to like the high salt concentration. Do duplicate plates. 
    Quality control on plates: be sure to streak a non-transformed plate every time to make sure the antibiotic is working.
    Transform into a hardier strain/one better suited for transformations?

    Project Goals

    1. Create a T7 Riboregulation System to control the expression of our proteins:
    We are dealing with anti-microbial peptides, so there is the possibility that the peptide we create would be toxic to E. coli which we are using to synthesize the peptide. We created a plasmid with specific locks in place so that we control when the T7 RNA polymerase, an RNA polymerase from the T7 bacteriophage, is expressed. Once the T7 RNA polymerase is expressed, it can then specifically target the T7 Promoter located in a different plasmid, which will lead to the expression of the specific peptide we want. (Show Figure 3)

    2. Design the anti-biofouling peptide using both a modular approach:
    In order to carry this out, we used the foot protein consensus sequence mefp 1-mgfp 5-mefp-1, which was found to be effective in Lee et al., 2008. At the N-terminus is the twin Strep-FLAG tag (using Strep tag for purification, and FLAG tag for easy cleavage). Then, the LL-37 antimicrobial peptide (AMPs are generally short enough to be inserted via primer overhang) is present on a long 36 residue linker. On the other side of the foot protein is sfGFP connected by a shorter linker. With targeted primers, the construct can be amplified in its entirety, or only with the AMP or GFP segment (Show Figure 6).

    3. Develop an erosion rig to assess the strength of the adhesive peptide:
    (Show figure 8) First, we will need to determine if we have adhered material present in various solutions and surfaces. In order to these this out, we will look at the contact angle measurement. Surfaces that are wet will have a very shallow contact angle because the surface absorbs the test liquid. Non-wetting surfaces will usually exhibit an obtuse contact angle because there is no absorption. This test will determine if our coating is present and does not dissolve when wet. As a further test to determine if the material is able to adhere to surfaces, we will use Fourier Transform Infrared Spectroscopy (FTIR). The adhesive should exhibit a different spectrum than uncured adhesive. This difference probably lies in the different vibrational bond energies caused by coordination or bonding to our surface. The next assessment will be to determine how much coating is retained under stress with atomic force microscopy (AFM). A probe will be applied to the sample to determine the force between the atoms of the sample and the atoms of the tip. Image contrast can then be generated by monitoring the forces of the interactions between the tip and the peptide’s surface.

    <img src="800px-Erosion_Rig_Image.png">

                   Figure 8. A diagram illustrating the configuration of the erosion rig developed to introduce adhesive coated surfaces to liquid erosion.<i>
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    Main Campus:
    Molecular, Cellular & Developmental Biology
    219 Prospect Street
    P.O. Box 208103
    New Haven, CT 06520
    Phone: 203.432.3783
    igem@yale.edu
    natalie.ma@yale.edu (Graduate Advisor)
    Copyright (c) 2014 Yale IGEM

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