Overview: Delivering Antibiotic Susceptibility in vivo

In order to modify a MRSA population and in turn, have the population able to propagate the modification, a method of delivery is essential. Conjugation, the horizontal transfer of genetic material between bacterial cells, is the proposed method for this delivery.

We decided to use conjugation as our mode of delivery because it has a large carrying capacity. Our silencing systems are quite large and therefore need the appropriate delivery mechanism. While a Staphylococcus virus would be more efficient at transferring DNA to a recipient cell, there are no Staphylococcus viruses that have been found with a carrying capacity large enough to handle our silencing systems.

Conjugation is a transfer of genetic material between prokaryotes by cell-to-cell contact. This process is well characterized in gram-negative bacteria, such as E. coli, but it is less characterized in gram-positive bacteria like Staphylococcus aureus. To infer information on the gram-positive process, microbiologists usually use the gram-negative process as a template (Grohmann et al, 2003).

The conjugation process in both gram-positive and gram-negative bacteria all share underlying similarities. For example, an origin of transfer (oriT) is required for all plasmid conjugation. A multiprotein complex that binds to the origin of transfer is called the relaxosome. The proteins involved in the relaxosome are coded within a tra/trs region that can be found on the plasmid or on the chromosome. The relaxosome is recognized by DNA relaxes which are able to perform a single or double stranded cut (depending on the type of system). The DNA released from the cut is then available to transfer to the recipient cells (Grohmann et al, 2003).

Design

Strain Considerations

To test our system in the lab, a Level 1 organism was used as a safety precaution. Staphylococcus epidermidis (ATCC 12228) is a close relative of Staphylococcus aureus and is able to conjugate with S. aureus populations (Forbes and Schaberg, 1983).

Conjugation Machinery

The plasmid pGO1 is of the few gram positive conjugative plasmids studied. The entire 54 kb plasmid has been sequenced by Caryl and O’Neill (2009) (Accession: NC 012547.1). The parts of the pGO1 conjugative plasmid sufficient for trans-species conjugation include a ~ 14 kb trs gene cluster, and a ~2 kb region containing the oriT and a gene which encodes for a nickase protein (Climo et al., 1996).

Experiment

In order to test the conjugation machinery in S. epidermidis, we sought to construct a conjugative plasmid containing the oriT region and trs region from pGO1. The plasmid should maintain a low copy number to ensure a lower metabolic load imposition which would increase the chance of acceptance by the cell. The low copy, theta replicating oriV from the Staphylococcal plasmid pSK41 (BBa_K1323018) was a great candidate. An erythromycin resistance gene (BBa_K1323011) was chosen as the selection marker and DsRed (BBa_K1323015) (Figure 1) was chosen as a reporter to track the movement of the plasmid.

DsRed fluorescence was analyzed in both E. coli and S. epidermidis. The DsRed cassette (BBa_K1323015) was incorporated into a pUC57 plasmid containing an erythromycin resistance gene (BBa_K1323011) and Staphylococcal oriV (BBa_K1323018). This plasmid was transformed into the strains using heat shock for E. coli and electroporation for S. epidermidis. Pellets from 50 ml overnight cultures were collected into 1.5 ml eppendorf tubes. To the naked eye (A) E. coli has a pink color (left), whereas in S. epidermidis (middle), a slight pink colour could only be noticed if compared to S. epidermidis without the construct (right). When seen under a RFP filter (B) a high degree of red fluorescence was observed in E. coli (left) and a lower degree of red fluorescence was seenin S. epidermidis (middle). The S. epidermidis negative control (right) showed no observable fluorescence.

Since construction of this plasmid would take place in E. coli, the parts would be cloned into the pSB1A3 backbone. The final conjugational plasmid to test in the lab is shown in Figure 2.

The design for the final test plasmid for conjugation. The plasmid contains the trs and nes regions from pGO1 which are essential for conjugation, a DsRed reporter gene (fluoresces red), and erythromycin resistance gene as a selective marker in Staphylococci, a low copy Staphylococci origin of replication, within the pSB1A3 backbone which contains a high copy E. coli origin of replication and ampicillin selection marker.

The pGO1 plasmid was graciously sent to us by Dr. Alex O’Neill’s lab at the University of Leeds. The region containing the oriT and nickase protein (position 8021-10264 on the pGO1 plasmid) was successfully PCR amplified, cloned into pSB1C3, and sent to the registry as a biobrick (BBa_K1323003). Amplification and cloning of the trs region (position 23723 - 37201 on the pGO1 plasmid) is still in progress. Once this conjugational plasmid is constructed, mating assays will be performed between S. epidermidis to ensure all parts are functional.

Design Considerations

The antibiotic resistance genes on the plasmid were used as a means to work with the constructs in the lab. However, they would be problematic if this design was implemented as a treatment for MRSA so we propose that these genes be removed in the final product