Difference between revisions of "Team:SDU-Denmark/Proof"
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<h3><strong>Bacteriocins – They work!</strong></h3> | <h3><strong>Bacteriocins – They work!</strong></h3> | ||
| − | <p | + | <p>In the project we designed biobricks, which contain the genes encoding a single bacteriocin or a hybrid bacteriocin. We purified the bacteriocins using the IMPACT method and determined the respective concentrations using a Bradford standard protein assay with Bovine Serum Albumin (BSA). |
| − | + | The first key criteria in order to purify the bacteriocins were to correctly incorporate the bacteriocin into the IMPACT vector pTXB1. The picture below shows the result of colony PCR products. Well 1-3 + 5-7 + 13-14 corresponds to the bacteriocin ThuricinS incorporated into the plasmid pTXB1, marked by the red box. The results show bands of similar length, which indicate a successful ligation between ThuricinS and pTXB1. Let the purification begin! </p> | |
| − | <p | + | <p><strong>Performing a MIC test tested the activity of the bacteriocins.</strong> Our MIC test shows growth inhibition of the strains MRSA:USA300 <em>(S.aureus)</em>, MRSA:CC398 <em>(S.aureus)</span></em>, hetero-VISA <em>(S.aureus)</em> and <em>Pseudomonas aeruginosa.</em> The bacteriocins LacticinQ and the hybrid Laterosporulin-ThuricinS did not elicit their activity towards <em>P. aeruginosa.</em> However the absence of inhibition could be due to a higher MIC value i.e. need of a higher concentration to inhibit growth, than used in the MIC test which still leaves the hybrid as a potential inhibitor of <em>P. aeruginosa</em></p> |
| − | <p><strong> Compared to traditional antibiotic the bacteriocins has similar or better effect </strong>. The bacteriocins shows inhibition of growth towards MRSA strains and P.aeruginosa at concentrations where the traditional antibiotics does not elicit an effect. The bacteriocins thus show promising results to support the idea of bacteriocins | + | <p><strong> Compared to traditional antibiotic the bacteriocins has similar or better effect </strong>. The bacteriocins shows inhibition of growth towards MRSA strains and <em>P. aeruginosa</em> at concentrations where the traditional antibiotics does not elicit an effect. The bacteriocins thus show promising results to support the idea of bacteriocins being a supplement for traditional antibiotics of which multiresistant bacterias has become less sensitive towards. </p> |
<p><strong>Synergistic effect of bacteriocins</strong></p> | <p><strong>Synergistic effect of bacteriocins</strong></p> | ||
| − | <p> | + | <p>The bacteriocins inhibit growth of the respective strains BUT the bacteriocins also showed synergistic effect. The synergistic effect is shown by a decrease in MIC when the bacteriocin Laterosporulin and ThuricinS are combined as a hybrid compared to their MIC value as single proteins. The hybrid LacticinQ-LacticinZ also shows a decreased MIC compared to LacticinQ as a single acting bacteriocin. The absence of inhibition towards<em> P. aeruginosa </em> by Laterosporulin-ThuricinS when in a hybrid, compared to their single protein effect could be stated as a loss of effect. However the MIC values overall decrease towards the<em> S. aureus</em>strains indicating a stronger effect. The observation leaves the possibility of a gain of effect towards strains not tested in the MIC test. The gain of effect could possible prove another characteristic of the designed hybrid bacteriocins. This leaves the opportunity to design an antimicrobial compound as needed.</span></p> |
| − | <p><strong>Our bacteriocins face AMR | + | <p><strong>Our bacteriocins face AMR </strong> </p> |
| + | <p>The bacteriocins could serve as a supplement for traditional antibiotics, which bacterias are less sensitive towards. In the case our purified bacteriocins inhibit the growth of methicillin resistant S. aureus strains. It could be hypothesized that a combination of traditional antibiotics with bacteriocins could elicit a synergistic effect towards resistant bacterial strains. This leave us with the opportunity to shift the balance of resistance and face the evolution of antimicrobial resistance (<a href="Dan, Y., M. P. Zacharof and R. W. Lovitt (2012). "Bacteriocins Produced by Lactic Acid Bacteria a Review Article." APCBEE Procedia 2: 50-56." target="_blank">Dan, Y., M. P. Zacharof and R. W. Lovitt (2012)</a>).</strong></p> | ||
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Revision as of 09:10, 13 October 2016
Proof of Concept
Bacteriocins – They work!
In the project we designed biobricks, which contain the genes encoding a single bacteriocin or a hybrid bacteriocin. We purified the bacteriocins using the IMPACT method and determined the respective concentrations using a Bradford standard protein assay with Bovine Serum Albumin (BSA). The first key criteria in order to purify the bacteriocins were to correctly incorporate the bacteriocin into the IMPACT vector pTXB1. The picture below shows the result of colony PCR products. Well 1-3 + 5-7 + 13-14 corresponds to the bacteriocin ThuricinS incorporated into the plasmid pTXB1, marked by the red box. The results show bands of similar length, which indicate a successful ligation between ThuricinS and pTXB1. Let the purification begin!
Performing a MIC test tested the activity of the bacteriocins. Our MIC test shows growth inhibition of the strains MRSA:USA300 (S.aureus), MRSA:CC398 (S.aureus), hetero-VISA (S.aureus) and Pseudomonas aeruginosa. The bacteriocins LacticinQ and the hybrid Laterosporulin-ThuricinS did not elicit their activity towards P. aeruginosa. However the absence of inhibition could be due to a higher MIC value i.e. need of a higher concentration to inhibit growth, than used in the MIC test which still leaves the hybrid as a potential inhibitor of P. aeruginosa
Compared to traditional antibiotic the bacteriocins has similar or better effect . The bacteriocins shows inhibition of growth towards MRSA strains and P. aeruginosa at concentrations where the traditional antibiotics does not elicit an effect. The bacteriocins thus show promising results to support the idea of bacteriocins being a supplement for traditional antibiotics of which multiresistant bacterias has become less sensitive towards.
Synergistic effect of bacteriocins
The bacteriocins inhibit growth of the respective strains BUT the bacteriocins also showed synergistic effect. The synergistic effect is shown by a decrease in MIC when the bacteriocin Laterosporulin and ThuricinS are combined as a hybrid compared to their MIC value as single proteins. The hybrid LacticinQ-LacticinZ also shows a decreased MIC compared to LacticinQ as a single acting bacteriocin. The absence of inhibition towards P. aeruginosa by Laterosporulin-ThuricinS when in a hybrid, compared to their single protein effect could be stated as a loss of effect. However the MIC values overall decrease towards the S. aureusstrains indicating a stronger effect. The observation leaves the possibility of a gain of effect towards strains not tested in the MIC test. The gain of effect could possible prove another characteristic of the designed hybrid bacteriocins. This leaves the opportunity to design an antimicrobial compound as needed.
Our bacteriocins face AMR
The bacteriocins could serve as a supplement for traditional antibiotics, which bacterias are less sensitive towards. In the case our purified bacteriocins inhibit the growth of methicillin resistant S. aureus strains. It could be hypothesized that a combination of traditional antibiotics with bacteriocins could elicit a synergistic effect towards resistant bacterial strains. This leave us with the opportunity to shift the balance of resistance and face the evolution of antimicrobial resistance (Dan, Y., M. P. Zacharof and R. W. Lovitt (2012)).
