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?
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.
Figure 8. A diagram illustrating the configuration of the erosion rig developed to introduce adhesive coated surfaces to liquid erosion.
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Phone: 203.432.3783
igem@yale.edu 
