GFP Expression Check

To verify the effectivity of the general schema for our DNA [ Promoters Long (BBa_I712074) and Short (BBa_I719005), 5’ UTR (BBa_K1758100), Terminator(BBa_B0012) ] we decided to test our system in a bacterial plasmid under T7 control via the IPTG inducible lac operon.

This experiment was conducted by first cloning plasmid components together. A G-block was synthesized by IDT containing 5’UTR, Superfolder GFP (BBa_I746916), and Terminator (BBa_B0012). These were then amplified with overhang PCR via primers containing promoter sequences for T7 polymerase, two separate promoters were tested: Long (BBa_I712074) and Short (BBa_I719005). The now full translational units were amplified again with gibson primers for insertion into pSB1C3 amplified with complimentary gibson primers. These linear amplicons were verified via gel electrophoresis, purified, and finally gibsoned together and transformed into NEB T7 express competent E. coli. The transformed colonies were exposed to broad spectrum UV radiation to excite GFP and phenotypically select for colonies with the correct construct. Colonies with correct expression were taken into liquid culture for larger scale verification of fluorescence and miniprep.

AMP Bacterial Killing by OD600 Broad Investigation

Functional killing of AMPs was tested in a broad format. Each of our Antimicrobial peptide candidates was constructed as a G-block which contained the 5'UTR, the AMP gene, and the Terminator. These were then amplified with tailed primers containing the promoter sequences for the short and long promoter respectively. These amplified constructs were verified via gel electrophoresis and purified and quantified via Nanodrop.

This experiment was designed to broadly test the bacteriostatic effect of each AMP under the control of each promoter and with or without the presence of the methionine aminopeptidase enzyme. Antimicrobial peptides were and no template controls were expressed over 4 hours at 37C using our in-house prepared cell-free reaction mixture. A culture of BL21 E. coli was raised overnight in LB and then diluted to apx 1*10^8 CFU/ml. 90ul of the dilute culture was combined with 10ul of each AMP reaction mixture and then placed in a plate reader shaking at 37C for several hours. Cell-free reaction mixtures that had no template DNA were also added to several wells as a growth control. OD600 was measured every 15 minutes.

AMP Bacterial Killing by OD600 Specific Investigation

The first bacterial killing assay had shown several AMP candidates which potentially had a bacteriostatic effect. To verify this effect in a more rigorously controlled experiment with replicates was conducted. The AMPs most consistent across promoter type and presence or absence of methionine aminopeptidase were: ISGCock_Contig05_0593 (BBa_K2431011), BP100 (BBa_K2431016), and Micrococcin C7 (BBa_K2431010). These AMPs and additional Methionine aminopeptidase (BBa_K2431018) were re-amplified from their respective G-blocks and subsequently purified using the same process detailed above. Antimicrobial peptides and no template controls were expressed in triplicate over 4 hours at 37C using our in-house prepared cell-free reaction mixture. A culture of BL21 E. coli was raised overnight in LB and then diluted to apx 1*10^8 CFU/ml. 90ul of the dilute culture was combined with 10ul of each AMP reaction mixture and then placed in a plate reader shaking at 37C for 18 hours. Cell-free reaction mixtures that had no template DNA were also added to several wells as a growth control. OD600 was measured every 15 minutes.

AMP Bacterial Killing by Plate using PURExpress

The goal of this experiment was to use the PURExpress system to produce the AMPs of potential effect in the initial broad OD600 investigation. Evaluate microbial killing effectivity of a droplet of the expression rxn on a BL21 bacterial lawn in order to check whether or not the in-house preparation of the cell free reaction mixture was effective at expressing AMPs. PURExpress reactions were prepared for BP100 (BBa_K2431016), and Micrococcin C7 (BBa_K2431010) with both promoter arrangements, a no DNA added reaction was also prepared as a control. These reactions proceeded at 37C for 3 hours for expression to occur. Reactions were then added to plates containing a lawn of BL21 E. coli. For each reaction 1 20ul droplet of undiluted reaction mixture and 1 20ul droplet of reaction mixture diluted 1:4 were added to separate plates.