Team:SDU-Denmark/Proof
Proof of Concept
Bacteriocins - They work!
During 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. We identified successful ligation between our bacteriocins and the pTXB1 with colony PCR, thus the first key criteria were achieved. Let the purification begin!
Purified bacteriocins inhibit the growth of MRSA and P. aeruginosa
Our MIC test shows growth inhibition of the strains MRSA:USA300, MRSA:CC398, hetero-VISA and P. aeruginosa:PAO1. 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. the 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.
Synergistic effect of bacteriocins
Figure 1 visualizes MIC values for each bacteriocin towards each tested strain. The respective strains are listed in the x-axis and the MIC values are plotted logarithmic(2) on the y-axis. The lowest bar indicates the bacteriocin eliciting the strongest effect.
Figure 1 visualizes MIC values for each bacteriocin towards each tested strain. The respective strains are listed in the x-axis and the MIC values are plotted logarithmic(2) on the y-axis. The lowest bar indicates the bacteriocin eliciting the strongest effect.
Compared to traditional antibiotics, our results show that the bacteriocins has similar or better effect. The bacteriocins show inhibition of growth towards MRSA strains and P. aeruginosa at concentrations were the traditional antibiotics does not elicit an effect. The bacteriocins therefore show promising results to support the idea of bacteriocins being a supplement for traditional antibiotics, of which multiresistant bacterias has become less sensitive towards.
The bacteriocins not only inhibited the growth of the tested strains BUT the bacteriocins also showed synergestic 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. aureus strains, thus 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.
A functional Secretion System
Top10/pSB1C3-PanK-Sec secretes visible aggregates of PHB.
We assembled the BioBrick K2018050, which consists of a hemolysin based secretion system and pantothenate kinase II. The BioBrick was ligated into pSB1C3 with 3A assembly, prior to testing. The effect of the BioBrick has been confirmed qualitatively through culture growth.
Since our bacterial strains can secrete plastic, PHB extraction and production can be performed in entirely new and innovative ways. Secretion of PHB allows us to retrieve plastic without using toxic and expensive chemicals and thus possibly even allow us to utilize a Flow Bio Reactor for continuous PHB production.
At this point we cannot confirm whether the BioBrick can produce more plastic than non-secreting PHB producing strains, as we have not received consistent results from parallel extraction experiments with secreting and non-secreting PHB producing strains. We have however, extracted essential amounts of PHB from top10/pSB1C3-PanK-sec in order to 3D print.
From Culture flask to 3D Printing
The designed BioBricks: (K2018022, K2018023, K2018026, K2018027, K2018028, K2018029, K2018049 and K2018050) contained the genes of accumulating PHB inside the cell and the genes of secreting PHB. The PHB was recovered by the extraction method with hypochlorite. We allowed PHB to dry overnight and the purified PHB was thereby ready to be processed into filaments. These filaments were used under real world conditions through 3D printing, as shown below.
