Difference between revisions of "Team:SDU-Denmark/Proof"

 
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<h3><strong>Bacteriocins &ndash; They work!</strong></h3>
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<h3><img src="https://static.igem.org/mediawiki/2016/e/e9/T--SDU-Denmark--minibacto.png" height="16px" style="display:inline-block;margin-right:5px;vertical-align:middle;">Bacteriocins - They work!</h3>
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<p><span style="font-weight: 400;">In the project we have created the biobricks, which contain the genes encoding our bacteriocins. We purified the bacteriocins using the IMPACT method and determined the respective concentrations using a Bradford standard protein assay with Bovine Serum Albumin (BSA) as a reference. In order to purify our bacteriocins, correct incorporation of the bacteriocin gene into the IMPACT vector pTXB1 were validated by cPCR.</span></p>
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<p>We designed BioBricks, which contain the genes encoding a single or a hybrid bacteriocin. We <a href="https://2016.igem.org/Team:SDU-Denmark/Demonstrate#purify" target="_blank">purified</a> the bacteriocins using the IMPACT method and determined the respective concentrations using a <a href="https://2016.igem.org/Team:SDU-Denmark/Demonstrate#determine" target="_blank">Bradford Standard Protein Assay</a> with Bovine Serum Albumin. In order to purify the bacteriocins we <a href="https://2016.igem.org/Team:SDU-Denmark/Demonstrate#purify" target="_blank">correctly incorporate</a> the bacteriocin into the IMPACT vector pTXB1.</p>
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<p><span style="font-weight: 400;">The picture below shows the result of colony PCR products</span><span style="font-weight: 400;">. </span><span style="font-weight: 400;">Well 1-3 + 5-7 + 13-14 corresponds to the bacteriocin Thuricin S incorporated into the plasmid pTXB1, marked by the red box in the above picture. The results show bands of similar length, thus verifying a successful ligation with Thuricin S inside the IMPACT vector pTXB1. Let the purification begin! </span></p>
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<p><span style="font-weight: 400;">Performing a MIC test tested the activity of the bacteriocins. Our MIC test shows inhibition of growth of the strains USA300 (</span><em><span style="font-weight: 400;">S.aureus</span></em><span style="font-weight: 400;">), CC398 (</span><em><span style="font-weight: 400;">S.aureus</span></em><span style="font-weight: 400;">), hetero-VISA (</span><em><span style="font-weight: 400;">S.aureus</span></em><span style="font-weight: 400;">) and </span><em><span style="font-weight: 400;">Pseudomonas aeruginosa.</span></em><span style="font-weight: 400;"> The bacteriocins Lacticin Q and the hybrid Laterosporulin-Thuricin S did not elicit their activity towards </span><em><span style="font-weight: 400;">P.aeruginosa. </span></em><span style="font-weight: 400;">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 </span><em><span style="font-weight: 400;">P.aeruginosa</span></em></p>
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<h5>Purified bacteriocins inhibit the growth of MRSA and <i>P. aeruginosa</i></h5>
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<p><strong>Synergistic effect of bacteriocins</strong></p>
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<p> Our MIC-tests showed <a href="https://2016.igem.org/Team:SDU-Denmark/Demonstrate#MIC" target="_blank">growth inhibition</a> of the strains MRSA:USA300, MRSA:CC398, hetero-VISA and <i>P. aeruginosa</i>:PAO1. The bacteriocins LacticinQ and the hybrid Laterosporulin-ThuricinS did not inhibit the growth of <em>P. aeruginosa.</em> However, the absence of inhibition could be due too low concentration used in the MIC test.</p>
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<p><span style="font-weight: 400;">Not only did the bacteriocins inhibit growth of the respective strains &ndash; the bacteriocins also showed synergistic effect. The synergistic effect is shown by a decrease in MIC when the bacteriocin Laterosporulin and Thuricin S are combined as a hybrid compared to their MIC value as single proteins. The hybrid LacticinQ-LacticinZ also shows a decreased MIC compared to Lacticin Q as a single acting bacteriocin. The absence of inhibition towards </span><em><span style="font-weight: 400;">Pseudonomas aeruginosa</span></em><span style="font-weight: 400;"> &nbsp;by Laterosporulin-Thuricin S when in a hybrid, compared to their single protein effect could be stated as a loss of effect. However the MIC values over all decrease towards the</span><em><span style="font-weight: 400;"> S.aureus </span></em><span style="font-weight: 400;">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 be tested by use of other strains than tested towards and possible prove another characteristic of the designed hybrid bacteriocins. This leaves the opportunity to design an antimicrobial compound as needed.</span></p>
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<p><strong>Our bacteriocins face AMR</strong></p>
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<br>
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<p><strong><span style="font-weight: 400;">The bacteriocins could serve as a supplement for traditional antibiotics, which bacterias has evolved resistance towards as 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 strains that have evolved resistance towards the antibiotics thus leaving 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). &quot;Bacteriocins Produced by Lactic Acid Bacteria a Review Article.&quot; APCBEE Procedia 2: 50-56." target="_blank">Dan, Y., M. P. Zacharof and R. W. Lovitt (2012)</a>).</span></strong></p>
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<h5>Synergistic effect of bacteriocins</h5>
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<a href="#demonstrate4"><img src="https://static.igem.org/mediawiki/2016/e/eb/T--SDU-Denmark--MICvisualdatacorrect.png" alt="" width="100%"></a>
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      <img src="https://static.igem.org/mediawiki/2016/e/eb/T--SDU-Denmark--MICvisualdatacorrect.png" alt="" width="100%">
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      <p>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.</p>
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<p  class="figuretext"><em>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.</em></p></div>
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<p>Our results showed that the bacteriocins have a <a href="https://2016.igem.org/Team:SDU-Denmark/Demonstrate#synergy" target="_blank">similar or better effect</a> compared to traditional antibiotics. The bacteriocins therefore show promising results to support the idea of bacteriocins being a supplement for traditional antibiotics.</p>
 +
<p>The bacteriocins did not only inhibit the growth of the tested strains but also showed <a href="https://2016.igem.org/Team:SDU-Denmark/Demonstrate#synergy" target="_blank">synergestic effect</a>. 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<i> P. aeruginosa </i> 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<i> S. aureus</i> 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.</p></div><br>
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<h3><img src="https://static.igem.org/mediawiki/2016/8/87/T--SDU-Denmark--miniPHB.png" height="16px" style="display:inline-block;margin-right:5px;vertical-align:middle;">A functional Plastic Secretion System</h3>
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  <img src=" https://static.igem.org/mediawiki/2016/e/ed/T--SDU-Denmark--plast_production.jpg" alt="" width="100%">
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  <p> <em>Figure 2: Top10/pSB1C3-PanK-Sec secretes visible aggregates of PHB.</em></p>
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<p class="figuretext"><em> Figure 2: Top10/pSB1C3-PanK-Sec secretes visible aggregates of PHB.</em></p></div>
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<p>We designed the BioBrick <a target="_blank" href="http://parts.igem.org/Part:BBa_K2018050">K2018050</a>, 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.</p><br>
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<p>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 <i>Flow Bio Reactor</i> for continuous PHB extraction.</p><br>
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<p>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.</p>
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<h5 style="clear:right;">From Culture flask to 3D printing </h5>
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<p>The designed BioBricks: (<a target="_blank" href="http://parts.igem.org/Part:BBa_K2018022">K2018022</a>,
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<a target="_blank" href="http://parts.igem.org/Part:BBa_K2018023">K2018023</a>,
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<a target="_blank" href="http://parts.igem.org/Part:BBa_K2018026">K2018026</a>,
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<a target="_blank" href="http://parts.igem.org/Part:BBa_K2018027">K2018027</a>,  
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<a target="_blank" href="http://parts.igem.org/Part:BBa_K2018028">K2018028</a>,
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<a target="_blank" href="http://parts.igem.org/Part:BBa_K2018029">K2018029</a>,
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<a target="_blank" href="http://parts.igem.org/Part:BBa_K2018049">K2018049</a> and
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<a target="_blank" href="http://parts.igem.org/Part:BBa_K2018050">K2018050</a>)
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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. </p>
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Latest revision as of 02:09, 20 October 2016

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
X

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

X

Figure 2: Top10/pSB1C3-PanK-Sec secretes visible aggregates of PHB.

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.