Overview bacteriocin results

Purification of the bacteriocins by the IMPACT method

Determination of bacteriocin protein concentration using Bradford Standard Protein assay

Inhibition of growth of S.aureus MRSA strains and P.aeruginosa detected by MIC

Synergestic effect of hybrid bacteriocins detected by MIC

Bacteriocin purification

Successful cloning of the bacteriocins

Cloning into the IMPACT vector pTXB1 were shown by gel electrophoresis. A successful transformation were verified by a colony PCR with specially designed primers for each bacteriocin. The results were verified according to the theoretical expected length of the bacteriocin with small overhangs (1-4 nucleotides) which is seen in the current figure.

FIGUR T--SDU-Denmark--cPCRthuricinS_bacteriocin.png INDSÆTTES

Figur tekst; Results of a colony PCR originating from respective colonies from transformation of K2018011/pTXB1/E.coli Top10, K2018012/pTXB1/E.coli Top10 and K2018014/pTXB1/E.coli Top10. From right; Well 1-3= K2018011, Well 4 = K2018014, Well 5-7 = K2018011, Well 8 = K2018014, Well 9-12 = K2018012, Well 13-14 = K201801

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[The above figure shows the result of colony PCR products originating from respective colonies from transformation of K2018011/pTXB1/E.coli Top10, K2018012/pTXB1/E.coli Top10 and K2018014/pTXB1/E.coli Top10. Well 1-3 + 5-7 + 13-14 corresponds to the bacteriocin K2018011/pTXB1/E.coli Top10 (Thuricin S), marked by the red box in the above picture. The results show bands of similar length, thus showing a successful ligation of Thuricin S into pTXB1.]

Determination of bacteriocin concentration

We purified the bacteriocins using the IMPACT method. Link her til ”Experiments”. To assay the concentration of our purified bacteriocins, we used a Bradford standard protein assay of known concentrations of BSA (Bovine serum albumine) to compare measured absorbance of our bacteriocin to the relative absorbance of known standard protein concentrations.

DROPDOWN START. The respective concentrations are calculated according to y = 0,0011x + 0,0202. The protein concentration (x) is calculated as x = (y-0,0202)/0,0011. The measured absorbance and the respective protein concentrations are shown in the table below in µg/m L and ng/mL.DROPDOWN SLUT

Bacteriocin effect towards S. Aureus MRSA and P. aeruginosa

To assay the effect of our purified bacteriocins we performed a MIC test following SOP0025_v_1.

The bacteriocins were dissolved by a two-fold dilution in water from well A-G having the largest concentration possible at column A. The maximal concentration is the half of the calculated concentration of the bacteriocin as the proteins were dissolved by 2-fold when the respective bacteria cultures were added. We used different MRSA strains to test the effect of our bacteriocins in order to state the possibility for our bacteriocins as substitute for traditional antibiotic of which the bacteria has evolved resistance towards. Following strains where tested:

 MRSA CC398 – a methicillin and tetracyclin resistant Staphylococcus aureus isolated from a patient, which have gained it from a pig. The MRSA CC398 is called pig-MRSA which is found in many pigs in the Scandinavian and is a huge contributor to the resistance development.

Hetero - VISA – a Staphylococcus aureus strain. The strain is capable of growing at vancomycin concentration of >4mg/L., thus have gained relative vancomycin resistance. This is due to thickened walls of the strain, which is suggested to catch the vancomycin before entry. Conly, J. M., & Johnston, B. L. (2002).

USA300 – the Staphylococcus aureus strain is a community associated MRSA and is a common cause of skin infections. http://mbio.asm.org/content/6/2/e00054-15.abstract

Pseudonomas aeruginosa – the strain is commonly spread at hospitals thus given rise to re-hospitalization of people with open wound infection, which we would like our bacteriocins to face.

The MIC plates were performed with respective concentrations. The concentrations in the table indicate final concentrations i.e. the concentration of the bacteriocin in the well after adding bacterial culture. Maximal concentration without dilution of bacterial culture is indicated in the table. An example of the bacteriocin K2018010/Laterosporulin concentration value plated in the MIC wells is shown below.

MIC test

To validate growth or inhibition we use background absorbance from dH2O+Mueller Hinton (MH) Media + 10 % deviation as a reference value for estimation of growth. Below is shown a MIC plate for the bacteriocin Laterosporulin. The picture states a visual inhibition of growth of the respective strains.

FIGUR T--SDU-Denmakr--MICresultsvisual_Bacteriocins.png INDSÆTTES

Figure text: Visual MIC test results of K2018010/ Thuricin S. Well A(1-10) contain 33,5 µg/mL as final maximal concentration. A 2-fold dilution is performed in well B-G(1-10). Well H(1-10) does not contain bacteriocin, but contain bacterial culture. Well A-H(11-12) is BLANK.

OD values of MIC

A MIC was estimated according to the lowest concentration inhibiting growth due to the reference OD value based on MH-Media, dH2O and devitation. The table below shows the respective MIC values according to the tested strains and the bacteriocins they were exposed to. N.I = No inhibition. Thus no inhibition were shown at the measured maximum concentrations.

The MIC values are visualized in the bar-chard below. The bacteriocin eliciting the strongest effect is indicated by the lowest bar – thus the lowest MIC value. Compared to MIC values of traditional antibiotics i.e. Ampicillin and Chloramphenicol the bacteriocins show similar effect. HOWEVER the bacteriocins also show better effect due to inhibition of growth of strains which the traditional antibiotics does not inhibit

 FIGUR T--SDU-Denmark--MICresults_bacteriocins.png INDSÆTTES 

In the above figure the lowest bar – thus the lowest MIC value indicates the bacteriocin eliciting the strongest effect. Thuricin S effect all of the strains at a concentration of 16,6 µg/mL. Lacticin Q shows to affect only the Staphylococcus aureus strains at a MIC value of 22,2 µg/mL. Laterosporulin shows inhibition of growth towards all of the strains with the lowest MIC being towards the Staphylococcus aureus strains, USA300 and MSSA CC398, with a MIC at 16,8 µg/mL. Even though the laterosporulin as a single bacteriocin effect Pseudomonas aeruginosa, the Laterosporulin-Thuricin S hybrid does not elicit effect towards P.aeruginosa. However it is seen that the MIC values towards hVISA, USA300 and CC398 (13,1 µg/mL, 13,1 µg/mL, 6,6 µg/mL respectively) are lower than the MIC value found in their single bacteriocin form (Thuricin S MIC 16,7 µg/mL and Laterosporulin MIC 16,8 µg/mL). This indicates a synergistically effect of hybrid bacteriocins compared to their single effect.

Lacticin Q shows no inhibition towards Pseudomonas aeruginosa. However the absence of inhibition could also be due to a higher MIC value i.e. need of a higher concentration to inhibit growth than used in the MIC test. The hybrid LacticinQ-LacticinZ is seen to inhibit the growth of all of the above strains with a MIC equal to 7,6 µg/mL against the S. Aureus strains and a MIC corresponding to 15,2 µg/mL towards P.Aeruginosa. In addition the MIC is decreased compared to the MIC estimated for LacticinQ alone (22,2 µg/mL). This could be due to the presence of LacticinZ. However LacticinZ is not tested as a single bacteriocin and a direct comparison cannot state a decreased MIC for LacticinZ but only a decreased MIC for LacticinQ. Two bacteriocins working in concert (a hybrid) seem to contribute to a decrease in MIC and thus a stronger effect indicating synergy. It should however be noted that the hybrid Laterosporulin-Thuricin S “looses” its effect towards Pseudomonas aeruginosa when in a hybrid, compared to their effect as single proteins.

Replicating MIC

The MIC plates were incubated for 28 hours with equal OD value for each bacteria strain, thus equal amount of bacteria were added. For replicating the MIC experiment the respective generation times of the bacterial strains used should be considered. The generation times could diverge between the bacterial strains thus representing different conditions, which is reflected in the OD measured. Therefore the OD value between the strains cannot be compared, but the MIC values can. Furthermore instead of using duplicates, multiple replications could have been made for specification of the OD value. Thereby instead of calculating a mean value a median could be calculated comparing more OD values. By use of duplicates pipette errors cannot be neglected. A new MIC test could therefore be performed with multiplicates.

Overview plastic

Separation of the different strains due to different promoter and ribosomal binding sites ahead of the phaCAB genes

Additional ribosomal binding site is responsible for the largest increase in PHB

PHB producing cells

In order to determine the effectiveness of our library of PHB producing cells, we’ve used flow cytometry to get a picture of how effective the different strains were at producing plastic.The below results are made with 4 biologic replicates of each strain. The strains were incubated for 28 hours before the measurements were made.

Tabel tekst [Table X shows the content of the biobrick constructs tested.] 

Tabel indsættes - T--SDU-Denmark--PHB_biobrickconstruct_Plastic.png

Figur indsættes -  T--SDU-Denmark--PHB_Flowcymetry_Plastic.png

Figur tekst [Figure x: The intensity of the strain is displayed on the x-axis and the amount of cells with the corresponding intensity is displayed on the y-axis. Cells of the different strains are displayed in different colors and the strains included in the figure are; x (blue), y (red) z (green)(...).]

DROPDOWN START The figure shows a clear separation of the different strains. This we would expect for strains with different promoter and ribosomal binding sites ahead of the phaCAB genes. We included the biobrick created by tokyo tech in 2009 (K934001) and one by imperial College (K1149051) in 2012 as references for our new constructs. The strain containing their construct is displayed in the green color, from figure x (above). The highest intensity and detection of largest amount of cells is shown for the blue construct in figure x. The above suggest that one of our construct (blue) has a stronger ability than either biobrick to produce plastic.  DROPDOWN SLUT

Figur insættes - T--SDU-Denmark--PBH_productionRedflourescence_plastic.png

Figur tekst [Figure x+1:On the x-axis the different strains are displayed. The promoter and RBS are marked with corresponding affinity by “s” = strong, “W” = weak and “MS”=medium. The y-axis display the average intensity on the flow cytometer detected by red fluorescence. The intensity of the red fluorescence are calculated due to a mean value of the intensities detected in figure x by the flow cytometry.]

Unlike our initial expectations, the results suggest that the strongest additional promoter does not produce the largest amount of plastic shown by a less detected intensity for the Pro(S)-RBS(MS) in the above figure. However the result is difficult to confirm due to missing qualitative data. According to the above it could be suggested that the concentration of the phaCAB proteins becomes too high and thus start forming inclusions which reduce the effectivity of PHB production which is reflected by the less intentity detected in the figure X.

Equally surprising is the fact that the additional ribosomal binding site is responsible for the largest increase in PHB. The upstream ribosomal binding site for the PhaCAB gene is not followed by a start-codon and as a result ribosome binding to this RBS will not be able to start transcription. The strong and weak ribosomal binding sites generate less plastic detected by lower intensity. We suggest that the results seen due to changes to the local concentration of ribosomes caused by the presence of an additional ribosomal binding site upstream from the PhaCAB genes.

The figure below shows the correlation, suggested to cause the above observation.

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Figure x+2: illustration of Figur med 3 RNA strenge, én med stærkt RBS, én med medium RBS og én med svagt RBS.

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·The strong RBS will bind the ribosome tightly, and since there is no start codon following the first RBS, the local ribosomal concentration is only changed slightly as each ribosome will be attached to the ribosome for a long time.

·For a medium RBS, the affinity will still be high enough for ribosomes to be attached to the RNA frequently, but due to the lower affinity, they will also detach more frequently, resulting in an increase in the local concentration of ribosomes.

· For the weak RBS the affinity will be to low for the local concentration of ribosomes to be effected. DROPDOWN END