During the initial planning process of our project, we decided to use an antimicrobial peptide called Sushi S1 which derives from the lipopolysaccharide (LPS)-binding region of Factor C of horseshoe crabs. Our literature research showed that Sushi S1 has one of the lowest minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) against gram-negative bacteria like E. coli in general [1] and our target pathogen Pseudomonas aeruginosa in particular [2]. By choosing a highly effective AMP, we want to make sure that as much bacteria as possible are killed upon delivery of the plasmid and induction of peptide expression.
In previous publications the functionality of Sushi S1 was only tested by applying the synthesized peptide to the bacteria [3]. It was shown that the peptide binds with high affinity to Lipopolysaccharides (LPS) on the surface of the outer membrane of gram-negative bacteria [4,5]. Since we have not found any papers trying to express Sushi S1 inside the targeted bacterium, we decided to first test what cellular localization of the peptide has the biggest impact on the bacterial growth.
We designed three versions of our plasmid allowing us to test the effect of Sushi S1 at three different locations: in the cytoplasm, the periplasm or the extracellular space. In the framework of iGEM we first focused on experiments with the gram-negative bacterium E. coli.
Build
Using the pET-22b(+) plasmid backbone we cloned three variants of Sushi S1:
Cytoplasmic: pelB signal sequence was deleted from plasmid backbone
Extracellular: insertion of heat-stable toxin II (HSTII) secretion sequence [7]
For cloning purposes and plasmid amplification we used E. coli Top 10. The nucleotide sequence of Sushi S1 was ordered as a synthesized G-block fragment from IDT. Our cloning strategy for the various plasmids consisted of a combination of restriction digest, Gibson assembly and Site Directed Mutagenesis (get more details about the cloning process in our Notebook).
Test
We performed growth curve experiments in E. coli BL21(DE3) and E. coli BL21(DE3)-pRARE2-LysS and measured OD600 over time after induction of Sushi S1 expression (read more about our growth curve experiments on the Results page). We compared the three localizations with the growth of two different controls (empty pET-22b(+) plasmid and non-induced cultures).
Figure 1 Expression of Sushi S1 fused to different localization sequences in E. coli BL21(DE3) OD600 values of bacterial cultures after peptide induction with 0.5 mM IPTG, cultured at 30°C and 200 rpm in LB-medium (n = 2). pelB-Sushi: periplasmic Sushi S1, HSTII-Sushi: extracellular Sushi S1, Sushi: cytoplasmic Sushi S1. Controls: empty pET-22b(+) plasmid and uninduced bacterial cultures.
Our initial findings revealed:
Sushi S1 expression inhibited bacterial growth for all three localization sequences at least to some degree.
The extracellular version HSTII‑Sushi S1 showed the strongest growth impairment.
Induction of the empty pET-22b(+) plasmid also impaired growth. Therefore, a new control was required.
To address the issue of a new control and to verify our results we switched to pET-22b(+) plasmids containing the fluorescent protein mCherry as negative control. At this point we also changed our medium from LB‑medium to Mueller-Hinton broth (MH medium), as further literature research showed that it is recommended for testing antimicrobial susceptibility [8].
Although the results for HSTII-Sushi S1 were reproducible and bacterial growth was significantly reduced compared to mCherry expression, we observed that OD600 values started to increase again after approximately 6 hours.
Figure 2 Expression of HSTII-Sushi S1 in E. coli BL21(DE3) OD600 values of bacterial cultures after peptide induction with 0.5 mM IPTG, cultured at 30°C and 200 rpm in MH-medium (n=2). Sushi: extracellular HSTII-Sushi S1. Controls: pET-22b(+) plasmid containing HSTII-mCherry sequence and uninduced bacterial cultures.
To test if we could eliminate this effect, we repeated the experiment in E. coli BL21(DE3) with pRARE2 LysS plasmid. In this strain, Sushi S1 expression inhibited growth for 24 h, but the mCherry control also showed strong growth inhibition.
Learn
From our expression experiments with Sushi S1 in E. coli we can draw two main conclusions:
Effectiveness of AMP delivery concept Our chosen AMP Sushi S1 reduced bacterial growth at a significant level when produced within the target organism. Although the bacteria started growing again after about 6 hours, we were able to show that the concept of delivering a plasmid encoding a potent AMP to the bacteria works.
Optimal peptide localization (signal peptide selection) The secreted form of Sushi S1 (HSTII-Sushi S1) showed the strongest impact on bacterial growth. This aligns with literature data, showing that Sushi S1 binds to Lipopolysaccharides (LPS) on outer bacterial membranes.
Based on these findings, we decided to focus on HSTII-Sushi S1 in all following expression experiments in E. coli.
Design
During the initial planning process of our project, we decided to use an antimicrobial peptide called Sushi S1 which derives from the lipopolysaccharide (LPS)-binding region of Factor C of horseshoe crabs. Our literature research showed that Sushi S1 has one of the lowest minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) against gram-negative bacteria like E. coli in general [1] and our target pathogen Pseudomonas aeruginosa in particular [2]. By choosing a highly effective AMP, we want to make sure that as much bacteria as possible are killed upon delivery of the plasmid and induction of peptide expression.
In previous publications the functionality of Sushi S1 was only tested by applying the synthesized peptide to the bacteria [3]. It was shown that the peptide binds with high affinity to Lipopolysaccharides (LPS) on the surface of the outer membrane of gram-negative bacteria [4,5]. Since we have not found any papers trying to express Sushi S1 inside the targeted bacterium, we decided to first test what cellular localization of the peptide has the biggest impact on the bacterial growth.
We designed three versions of our plasmid allowing us to test the effect of Sushi S1 at three different locations: in the cytoplasm, the periplasm or the extracellular space. In the framework of iGEM we first focused on experiments with the gram-negative bacterium E. coli.
Build
Using the pET-22b(+) plasmid backbone we cloned three variants of Sushi S1:
Cytoplasmic: pelB signal sequence was deleted from plasmid backbone
Extracellular: insertion of heat-stable toxin II (HSTII) secretion sequence [7]
For cloning purposes and plasmid amplification we used E. coli Top 10. The nucleotide sequence of Sushi S1 was ordered as a synthesized G-block fragment from IDT. Our cloning strategy for the various plasmids consisted of a combination of restriction digest, Gibson assembly and Site Directed Mutagenesis (get more details about the cloning process in our Notebook).
Test
We performed growth curve experiments in E. coli BL21(DE3) and E. coli BL21(DE3)-pRARE2-LysS and measured OD600 over time after induction of Sushi S1 expression (read more about our growth curve experiments on the Results page). We compared the three localizations with the growth of two different controls (empty pET-22b(+) plasmid and non-induced cultures).
Figure 1 Expression of Sushi S1 fused to different localization sequences in E. coli BL21(DE3) OD600 values of bacterial cultures after peptide induction with 0.5 mM IPTG, cultured at 30°C and 200 rpm in LB-medium (n = 2). pelB-Sushi: periplasmic Sushi S1, HSTII-Sushi: extracellular Sushi S1, Sushi: cytoplasmic Sushi S1. Controls: empty pET-22b(+) plasmid and uninduced bacterial cultures.
Our initial findings revealed:
Sushi S1 expression inhibited bacterial growth for all three localization sequences at least to some degree.
The extracellular version HSTII‑Sushi S1 showed the strongest growth impairment.
Induction of the empty pET-22b(+) plasmid also impaired growth. Therefore, a new control was required.
To address the issue of a new control and to verify our results we switched to pET-22b(+) plasmids containing the fluorescent protein mCherry as negative control. At this point we also changed our medium from LB‑medium to Mueller-Hinton broth (MH medium), as further literature research showed that it is recommended for testing antimicrobial susceptibility [8].
Although the results for HSTII-Sushi S1 were reproducible and bacterial growth was significantly reduced compared to mCherry expression, we observed that OD600 values started to increase again after approximately 6 hours.
Figure 2 Expression of HSTII-Sushi S1 in E. coli BL21(DE3) OD600 values of bacterial cultures after peptide induction with 0.5 mM IPTG, cultured at 30°C and 200 rpm in MH-medium (n=2). Sushi: extracellular HSTII-Sushi S1. Controls: pET-22b(+) plasmid containing HSTII-mCherry sequence and uninduced bacterial cultures.
To test if we could eliminate this effect, we repeated the experiment in E. coli BL21(DE3) with pRARE2 LysS plasmid. In this strain, Sushi S1 expression inhibited growth for 24 h, but the mCherry control also showed strong growth inhibition.
Learn
From our expression experiments with Sushi S1 in E. coli we can draw two main conclusions:
Effectiveness of AMP delivery concept Our chosen AMP Sushi S1 reduced bacterial growth at a significant level when produced within the target organism. Although the bacteria started growing again after about 6 hours, we were able to show that the concept of delivering a plasmid encoding a potent AMP to the bacteria works.
Optimal peptide localization (signal peptide selection) The secreted form of Sushi S1 (HSTII-Sushi S1) showed the strongest impact on bacterial growth. This aligns with literature data, showing that Sushi S1 binds to Lipopolysaccharides (LPS) on outer bacterial membranes.
Based on these findings, we decided to focus on HSTII-Sushi S1 in all following expression experiments in E. coli.
References
[1] Li P, Sun M, Wohland T, Yang D, Ho B, Ding JL. Molecular Mechanisms that Govern the Specificity of Sushi Peptides for Gram-Negative Bacterial Membrane Lipids. Biochemistry. 2006 Sep 1;45(35):10554–62.
