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.
Our Solution
The Solution: To address this issue, we aimed to develop an anti-microbial peptide composed of two components, which we envision can be modulated to suit a variety of functional adhesive applications. The ideal anti-microbial peptide coating would exhibit both biofilm-inhibitory activity as well as strong adhesion to a variety of substrates. In addition, we sought to identify a biomimetic adhesive domain so as to enhance the environmental friendliness of our peptide. Mussel adhesive proteins (MAPs), which are secreted by the mussel to help it anchor and survive in the harsh conditions of the intertidal zone, are an effective and environmentally sound adhesive domain. As our anti-microbial domain, we selected LL-37, a member of the cathelicidin family of peptides, due to the potency of its toroidal pore mechanism of lipid bilayer disruption. MAPs selectively attach to inorganic and organic surfaces via L-dopamine (L-DOPA), which is generated by post-translational modification of tyrosine with tyrosinase (often secreted along with the MAPs by the mussels in an enzyme cocktail that assists with the crosslinking of MAPs). Our study sought to be the first to synthesize an entire recombinant anti-microbial adhesive peptide without the need for post-translational modifications. We incorporated L-DOPA, a nonstandard amino acid, into our construct using a genetically recoded organism (GRO). Because this peptide is toxic to the GRO in which it is produced, we designed a better controlled inducible system that limits basal expression. This was achieved through a novel T7 riboregulation system that controls expression at both the transcriptional and translational levels. This improved system is a precise synthetic switch for the expression of cytotoxic substances in the already robust T7 system. Lastly, a variety of tests were carried out to characterize our recombinant protein in comparison to the commercially available MAP-based adhesive, Cell-TakTM.
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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.
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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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Phone: 203.432.3783
igem@yale.edu 
