Team:SDU-Denmark/Proof
Proof of Concept
Bacteriocins - They work!
We designed BioBricks, which contain the genes encoding a single 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. In order to purify the bacteriocins we correctly incorporate the bacteriocin into the IMPACT vector pTXB1.
Purified bacteriocins inhibit the growth of MRSA and P. aeruginosa
Our MIC-tests showed 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 inhibit the growth of P. aeruginosa. However, the absence of inhibition could be due too low concentration used in the MIC test.
Synergistic effect of bacteriocins
Figure 1: visualizes MIC values for each bacteriocin towards each tested strain. The respective strains are listed on 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 on 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.
Our results showed that the bacteriocins have a similar or better effect compared to traditional antibiotics. The bacteriocins therefore show promising results to support the idea of bacteriocins being a supplement for traditional antibiotics.
The bacteriocins did not only inhibit the growth of the tested strains but also showed synergestic effect. This is indicated by the decrease in MIC when Laterosporulin and ThuricinS are combined as a hybrid compared to each respective MIC value. The hybrid LacticinQ-LacticinZ also showed a decreased MIC compared to the value of LacticinQ. The absence of inhibition towards P. aeruginosa by the hybrid Laterosporulin-ThuricinS compared to their single protein effect, could be due to a loss of function. However, the hybrid MIC values decreased towards the S. aureus strains, indicating a synergistic effect. The tendency for increased inhibition when bacteriocins are in a hybrid construct leaves the possibility of other strains similarly being inhibited, when exposed to a hybrid bacteriocin. Thus proving an important characteristic of the designed hybrid bacteriocin leaving the opportunity to design a synergistic antimicrobial compound.
A functional Plastic Secretion System
Figure 2: Top10/pSB1C3-PanK-Sec secretes visible aggregates of PHB.
We designed the BioBrick K2018050, consisting of a hemolysin based secretion system and pantothenate kinase II. The BioBrick was fused into the iGEM standard plasmid pSB1C3 via 3A assembly. The functionality of this construct has been qualitatively indicated through culture growth, see figure 2.
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 allowing us the utilization of a Flow Bio Reactor for continuous PHB extraction.
At this point we can not 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 non- and secreting PHB producing strains. We have however, extracted substantial amounts of PHB from top10/pSB1C3-PanK-sec in order to assess the possibility of its use in 3D printing.
From Culture flask to 3D printing
The designed BioBricks: (K2018022, K2018023, K2018026, K2018027, K2018028, K2018029, K2018049 and K2018050) containes the genes for accumulation of PHB inside the cell and the genes of the secretion system. The PHB was recovered by the hypochlorite extraction method. Where we allowed PHB to dry overnight. The purified PHB was processed into filaments and used under real life conditions in 3D printing, as shown below.
