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Description

As the name suggests, we decided to focus our project on attempting to inhibit biofilm formation via the alteration of genes in the quorum-sensing pathway found in Pseudomonas Putida.

Project description:

Biofilms are microbial communities living attached to a solid support such as catheters which are involved with 60% nosocomial infections (hospital acquired infections). This project would be the basis for preventing gram negative bacteria role in nosocomial infections by preventing the formation of these biofilm formation. Therefore, by generating a BioBrick part which can upregulate RasL and therefore downregulate quorum sensing intercellular combination of bacteria, can prevent bacterial communication and thus biofilm formation. This can benefit public health as gram-negative bacteria can cause a range of nosocomial infections including sepsis and pneumonia.

This project can help towards targeting antibiotic resistance. It is known that gram negative bacteria such as Pseudomonas aeruginosa and Pseudomonas Putida harbour multiple resistance to antibiotics (MDR). Specifically, biofilm-associated microorganism’s exhibit decreased susceptibility to antimicrobial agents. Susceptibility can be intrinsic, which is due to the growth in the biofilm. Susceptibility can also be extrinsic, which is due to the transfer of extrachromosomal elements to susceptible organisms in the biofilm. Therefore, having a target based intervention of the formation of the biofilm can prevent the decrease in the susceptibility of antimicrobials in a health care setting.

Application:

The implications of this project are vast in the public health sector. It can have direct effects on public health, using the BioPrick part generated to downregulate quorum sensing can be utilised in biological technology to directly prevent the adverse effects of biofilm formation such as infections which can cause a high fatality rate in patients who are immunocompromised for example. This project can also indirectly prevent the adverse effects such as costs and in fact improve infection control. Many problems associated with nosocomial infections include longer hospital stays, therefore increase in bed occupancy thus decrease in hospital space for new incoming patients, and ward closure, pain and anxiety for patients and families, loss of earning. Consequently, this project allows the universal approach in improving public health in the long term.

Characterisation of an existing biobrick part

Following on from initial experiments carried out by last year by Dr Anatoliy Markiv to compare the expression of pSB1c3 BBa_J04450 (RFP) from two different E. Coli competent cell strains: Xl1-Blue and TOP 10, we decided to broaden the experimental horizon with the inclusion of pSB1c3 BBa_K592101 (YFP) and DH5α competent cells. We studied a paper by Dvorak et al., (2015) on the toxicity of Isopropyl β-D-1-thiogalactopyranoside (IPTG) on the host bacterial cell and decided to study the effects of different IPTG concentrations on expression as well.

We set up eleven universal bottles containing LB broth for our experiments and added contents as seen in table 1 and figure 1:

Table 1: Preparation of LB broth contents

Figure1. Qualitative results from the experiments from table 1

We found that to better assess and interpret results, we should measure fluorescence in terms of expression which can be seen in figure 2 and 3.

Figure 2. Results for red fluorescent protein, Excitation: 585nm and Emission 610nm.

The results from figure 2 suggest that Tube 1 emits the greatest amount of fluorescence while tube 2 (with 0.1 mM IPTG) has repressed growth. This suggests that while TOP 10 does not have a lac inhibitor, the pSB1C3 backbone implanted allows control of expression, even if it is a high expression plasmid. DH5α expressed much lower rates of expression however 0.1mM IPTG increased expression rates. The results from the co-cultures suggest that without IPTG RFP could be expressed at a fairly medium rate however the addition of IPTG likely led to an overexpression of TOP 10 bacterial cells which led to overgrowth and cell death. This suggest that TOP 10 can heavily outgrow DH5α. Tube 10 contained pSB1c3 with BBa_Jo4450 however no antibiotic was added which suggested that the bacteria largely removed the plasmid.

Figure 3. Results for yellow fluorescent protein, Excitation: 510nm and Emission 530nm.

The results from figure 3 appear inconclusive as column 11 containing negative control gave positive results rendering the rest of the results unreliable. However, tube 5 containing pSB1c3 and part BBa_K592101 expressed higher amounts of YFP when compared to tube 6 in which IPTG was also added. This is similar to the results seen from tube 1 and 2, all 4 tubes inoculated with TOP 10 strains.