Plasmid Design

We decided to use the vector pUCP18 as a starting point to build our plasmid. It contains the origin of replication pRO1600 oriV BBa_K4083013 which originates from Pseudomonas aeruginosa POA and enables plasmid amplification in various Gram-negative bacteria [1]. Additionally, a high copy number origin of replication, ColE1 BBa_K4278506, is included in the backbone. This allows efficient plasmid replication and maintenance in E. coli [2], facilitating cloning procedures and plasmid manipulation.

The kanamycin resistance gene (KanR, BBa_K4284010), serves as a selection marker in our vector prototype, enabling cloning and testing in the lab. However, it can easily be removed via restriction digest, when no longer needed. After removing the selection marker and ligating the backbone, the finalized Pseudomonas specific plasmid is obtained.

The promoter is the most important building block to restrict antimicrobial peptide production to the targeted bacteria. CAPTURE uses a mutated version of the P. aeruginosa specific pel promoter.

Endogenously, the pel promoter controls the formation of biofilm dependent on the transcription factor FleQ and the second messenger cyclic diguanylate (c-di-GMP) [3]. This transcription system functions as a switch between the sessile and motile lifestyle of P. aeruginosa. Upon low levels of c-di-GMP, the FleQ/FleN transcription factor complex represses expression of biofilm associated genes, promoting the motile lifestyle of cells. At high concentrations, c-di-GMP binds to FleQ and causes a conformational change of the protein-DNA complex, which activates the transcription of genes responsible for exopolysaccharides production required for biofilm formation [4]. The bacterium switches to a sessile lifestyle.

Regulation of pel and FleQ activity can be uncoupled from the c-di-GMP level by introducing mutations into the protein binding sequences of the promoter. There are two regions of the pel promoter that can be bound by FleQ. Binding of FleQ to box 1 promotes biofilm formation, however, the binding of box 2 represses the transcription of biofilm related genes. Mutation of the promoter sequence in box 2 prevents binding of FleQ and as a result increases the transcription levels of biofilm associated genes. Expression activity analysis showed high transcriptional levels even at low c-di-GMP concentrations [5], allowing for constitutive expression of a gene of interest, largely unaffected by cellular c-di-GMP levels.

Homologs of the pel promoter and FleQ transcription regulator are also present in other Pseudomonas strains. After aligning the sequences of both components in P. aeruginosa and P. fluorescens we could see that the binding regions in promoter and protein are conserved, concluding that the system should be functional in our model organism as well.

In addition to the constitutive pel promoter, we also needed an inducible promoter for testing in the lab. We settled on the natural, Pseudomonas specific and xylose-inducible P_xut promoter, which is already characterized in Pseudomonas aeruginosa as well as in Pseudomonas fluorescens [7].

To stop gene expression on our plasmid we are using the strong terminator LUZ7 T50 BBa_K4757999 which has already been proven to function in both P. fluorescens and P. aeruginosa [8].

The heart of the plasmid is the gene encoding an antimicrobial peptide which is produced by the target pathogen Pseudomonas aeruginosa, eventually leading to the death of the bacterial cell.

We were looking for a preferably highly potent peptide, ensuring efficient lysis of as many bacteria as possible upon delivery of a plasmid. Eventually, we decided on the antimicrobial peptide Sushi S1, BBa_K5057004 derived from the lipopolysaccharide (LPS)-binding region of Factor C from the horseshoe crab. It shows high activity against Gram-negative bacteria in general [9], and strong effectiveness against Pseudomonas aeruginosa in particular [10].

Sushi S1 binds with high affinity to LPS on the surface of bacteria, leading to bacterial membrane disruption, causing cell leakage and death (get more information about AMPs in our Project Description) [11,12]. To enhance its effectiveness, we fused the signal peptide of the Poly(3-hydroxyoctanoate) depolymerase (PhaZ) to the N-terminus of Sushi S1, facilitating its secretion from the bacterial cell [13]. It is important to note that the PhaZ signal peptide is specific to P. fluorescens and is suitable only for initial testing in the model organism. For the final P. aeruginosa plasmid, we plan to use a different localization signal, such as the signal peptide of the P. aeruginosa produced extracellular elastase [14]. This adjustment ensures proper secretion of the AMP in the target pathogen.

We used the pET-22b(+) vector as the foundation for our E. coli-specific plasmid. The backbone contains the origin of replication ColE1 (BBa_K4278506) which allows plasmid amplification in E. coli.

Protein expression is controlled by the T7 promoter BBa_K4618093 which is regulated by the lac operon. Therefore, the sequences for the lac-operator BBa_K3286004 as well as the lacI repressor BBa_K2308015 and accordingly the lacI promoter BBa_K2572009 are encoded on the plasmid. Gene expression is induced in the presence of Isopropyl-β-D-thiogalactopyranoside (IPTG), making protein synthesis controllable. During our experiments, we observed that protein expression occurred even in the absence of the inducer, suggesting a relatively low leakiness of the promoter. Consistent with the promoter, the T7 terminator BBa_K3137010 regulates the stop of protein transcription. For selection, we used the antibiotic resistance gene AmpR BBa_K4696011, already present on the pET-22b(+) vector.

We designed several versions of the AMP construct to test different cellular localization. To examine the effectiveness of the different versions we fused Sushi S1 either to the signal peptide from E. coli enterotoxin heat-stable toxin II (HSTII, BBa_K5057002) for extracellular targeting or the pelB localization sequence BBa_J32015 for periplasmic localization. Additionally, we analyzed the effect of a cytoplasmically localized peptide expressed without a signaling sequence.

In addition to Sushi 1, we designed plasmids encoding another AMP called Conga BBa_K5057005. Conga is a synthetic peptide originally designed and tested in its D-amino acid configuration (D-CONGA-Q7), which has shown high potency against P. aeruginosa [15]. Although the activity of the L-enantiomer has never been tested, we decided to express the L-version of Conga in E. coli and analyze its effect compared to Sushi S1. Similar to our approach with Sushi S1, we designed two versions of the peptide: cytoplasmic (without localization sequence) and extracellular (fused to the HSTII signal sequence, BBa_K5057002).

Our eCPX-SpyTag encoding plasmid (see more at our integrated human practices) based on a pTrc99a backbone containing the high copy ColE1 origin of replication BBa_K2796022 alongside an Ampicillin resistance coding region consisting of an AmpR promoter BBa_K4696011 and a gene coding for Beta-Lactamase BBa_K4696011.

The expression of outer membrane proteins typically overloads protein translocation machinery, activating stress responses and leading to decreases in cell growth [17]. We designed two plasmids where we replaced the original inducible promoter with weak constitutive promoters: LacIq BBa_K3257003 and AmpR BBa_K2569033, respectively. The rrnB T1 terminator BBa_K3033016 and rrnB T2 terminator BBa_K2936015 are used for expression termination.

The expression of outer membrane proteins typically overloads protein translocation machinery, activating stress responses and leading to decreases in cell growth [17]. We designed two plasmids where we replaced the original inducible promoter with weak constitutive promoters: LacIq BBa_K3257003 and AmpR BBa_K2569033, respectively. The rrnB T1 terminator BBa_K3033016 and rrnB T2 terminator BBa_K2936015 are used for expression termination.

We use this backbone for the expression of SpyTag-eCPX BBa_K5057010. eCPX or the enhanced circularly permuted OmpX, is a recombinant outer membrane protein capable of expressing peptides on both the N- and C- termini [18]. We used it to express the SpyTag peptide on the surface of outer membrane vesicles that can be “caught” by proteins fused to a SpyCatcher in order to functionalise OMVs with targeting ligands.

We received the SpyCatcher encoding backbone, pBbA2c, from the 2022 iGEM Freiburg team. The plasmid contained the medium copy p15A ori BBa_K3982024 alongside a Chloramphenicol resistance coding region BBa_K3170994.

SpyCatcher expression was driven by the doxycycline inducible tetA/tetR promoter BBa_K2800025 and terminated via the endogenous rrnB T1 BBa_K3033016 and T7TE BBa_K3257024 terminators.

This plasmid was used for the expression and purification of a fusion protein consisting of SpyCatcher (a protein that forms a covalent bond with SpyTag), P2 phage-tail protein derived from the PRD1 bacteriophage [19] and a 6xHisTag BBa_K5057011 for purification purposes. The purified fusion protein could then be used to functionalize OMVs exhibiting a SpyTag.