Overview

Bacteriocin results --> her skal være et hyperlink. Når der klikkes springes ned til bacteriocin afsnit.

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

Constructing silk fibers --> her skal være et hyperlink. Når der klikkes springes ned til silke afsnit.

The MaSp1 and MaSp2 gene blocks were successfully inserted into a vector

It was possible to proof the concept of preparing silk from the iterative capped assembly method for one monomer of MaSp1 and MaSp2, each consisting of the gene fragments AB, BC and CA.

The gene blocks ligated as expected with the oligos and with each other

We did not manage to ligate the full construct due to use of expired streptavidin beads

Production optimization of plastic producing cells--> her skal være et hyperlink. Når der klikkes springes ned til plastik afsnit.

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

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

Sættes som dropdown el. bagved figur

[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.

BACTERIOCINS	OD  (595 nm)	µg/mL
IMPACT ELUAT (Laterosporulin-Thuricin S)	0,078	52,55
IMPACT ELUAT (Lacticin Q)	0,069	44,36
IMPACT ELUAT (Laterosporulin)	0,094	67,09
IMPACT ELUAT (Thuricin S)	0,057	33,45
IMPACT ELUAT (LacticinQ-LacticinZ)	0,0536	30,36

 

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.

Parts nr.	Bacteriocin/MIC [µg/mL]	hVISA	USA300	CC398	P.Aeruginosa
K2018011	Thuricin S	16,7	16,7	16,7	16,7
K2018012	Lacticin Q	22,2	22,2	22,2	N.I < 22,2
K2018010	Laterosporulin	33,6	16,8	16,8	33,6
K2018015	LacticinQ-LacticinZ	7,6	7,6	7,6	15,2
K2018014	Laterosporulin-Thuricin S	13,1	13,1	6,6	N.I <13,1

Traditional Antibiotic/MIC [µg/mL]	hVISA	USA300	CC398	P.Aeruginosa
Ampicilin	< 16,0	< 16,0	1000,0	N.I<1000,0
Chloramphenicol	78,0	N.I.<1000	N.I.<1000	< 39,0

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.

Silk preparation by Iterative Capped Assembly  (ICA)

Insertion of MaSp gene blocks into plasmid

The MaSp genes contain 3 fragments which we call “minimers”. The minimers together make up a monomer and thus a functional MaSp protein. Assembly of the minimers are essential for the construct of a functional silk fiber. The gene fragments were successfully inserted into the plasmid pSB1C3 and the ligated product was transformed into E. coli. The result from colony PCR appeared to be 400 bp. However it would have been expected around 433 bp (Figure 1). The lower bands could be due to uncertainties in the gel electrophoresis.

Colonies from the above cPCR were set for incubation overnight following plasmid purification. The purified plasmids were tested by digesting with the restriction enzymes EcoRI and PstI. The digested gene is represented by a band of 160 bp, corresponding to the size of one silk gene. The results were confirmed by DNA sequencing.

Preparation of genes for ICA

We used the original pPCR product from the genes as a template. We expected to see a band of 160 bp because of the added prefix and suffix. The primers used attach themselves to the prefix and suffix, and are thus included in the amount of base pairs after PCR. The event makes the restriction site of Eco31I more accessible for the enzyme. (Figure 3).

Indsæt Figur 3

Figure tekst [Figure 3: shows the results of a phusion PCR from a pPCR product template.

After digestion the pPCR product with Eco31I, bands at 106 bp were expected (Figure 4).

Indsæt Figur 4

Figur tekst [Figure 4: Digested pPCR product where the top band is our digested gene product and the lower band is a mix of primers and cut off parts of the gene product.]

ICA technique allows us to ligate different pieces of DNA together stepwise due to correct overhangs. We wanted to create the hybrid silk of a bacteriocin fused with a monomer of MaSp1, MaSp2 or even a four monomer with different proportions of the genes on DNA level. Eco31I recognizes four nucleotides but cuts four randomly base pairs at the location +1bp  upstream from recognition site. We were thus able to manipulate these base pairs to create a “MaSp1- and MaSp2 CD sequence ”, which would be specific for the recognitions sites of our bacteriocins. This feature still allows us to digest with the same restriction enzyme, Eco31I. Creation of these new gene sequences would enable us to determine, through ICA, where to incorporate the bacteriocins into the spider silk gene sequence, thereby forming a hybrid spider silk. The MaSp1 and MaSp2 CD sequence only differ in the overhang.

We tested the gene's’ ability to be ligated by a ligation test. The digested gene is 106 bp. A bond at 212 bp verified that the gene fragments can be ligated together (Figure 5).

Indsæt figur 5

Figur tekst [Figure 5: Ligation test. Bands at 106 bp are the unligated silk gene. Bands at 212 bp represent the ligated product, and thereby verifies a successful ligation test.]

It was possible to proof the concept of preparing silk from the iterative capped assembly method. One monomer of MaSp1 and MaSp2 was made by the ICA method (Figure X). One monomer of a MaSp gene consists of the gene fragments AB, BC and CA, and it is expected to have a length of 406 bp.

Figur indsættes - T--SDU-Denmark--ICAmonomer_MaSp1_and_MaSp2.png

Figur tekst [Figure x: ICA monomer of MaSp1 and MaSp2]

We tried to make constructs containing 4 MaSp gene monomers. However, we struggled succeeding in making a longer construct. The closest result we got was to make a gene containing 700 bp. To this point we can therefore only conclude that it was not possible to use the ICA method for longer constructs of MaSp genes, even though adjustments were made during the experiments. There is great potential in the method, but it should be considered whether the method is applicable for longer constructs.

The dream of creating a silk-bacteriocin hybrid

The idea was to incorporate bacteriocins between silk monomers thereby creating a hybrid silk fibre. From the literature it is known that silk doesn’t lose its function when other proteins are combined with the silk. We also found that proteins incorporated in silk monomers doesn’t lose their functions either, which why we suggests that the bacteriocins incorporated into silk monomers both would maintain their functions and thereby their effects. Our hypothesis is that silke fibres which is proven to be immune neutral and promotes wound healing, combined with the antimicrobial effect from the bacteriocins, would create a synergistic effect resulting in decreasing infections and also a decrease in healing time in patients with severe wounds. We wanted to fusion proteins on DNA level by using recombinant DNA technology, two methods was tested: Iterative Capped Assembly, and restrictions cloning. --> For further information check experiment --> link hertil.

We managed to prepare a bacteriocin with specific overhang matching the overhangs present on the MaSp genes in order to make them compatible. Lane no. 5 shows the bacteriocin Thuricin S with DA-overhangs. The band is expected to be 217 bpFermentas Generuler 50 kb ladder plus was used and the figure indicate that we succeeded at getting silk-overhangs on Thuricin S. For the other overhangs check out protocols: Thuricin S with silk-overhangs under results and conclusions.

Insæt figur m. bacteriocin overhang - T--SDU-Denmark-Bacteriocinoverhang_silk.png

Despite various attempts we never succeeded in cutting and ligate our Thuricin S together with a silk monomer. However we succeded in creating a bacteriocin with overhangs that matched the overhangs on the MaSp monomers, which in theory make it possible to combine the two fragments (results can be seen in the protocols). The overhangs is made to fit into our AB system, and Thuricin S thus have DA, DE, ED and EA overhangs. This combination of overhang give it potential to combine silk and bacteriocin in various ways. we can conclude that it all fit will in theory, but when it came to reality - it were difficult to create in practise. The bacteriocin with specific silk overhangs are therefor left to further research.

What went wrong - what to do?

ICA troubleshooting

Due to ICA’s multiple ligation steps, individual ligation test must be made in order to establish the viability of the constructs. Individual ligations between genes with matching overhangs are necessary, as the ICA method is time consuming where a singular failed ligation, can create negative results without knowing where the problem is. Ligation test includes the oligos Initiator, Terminator, A-Cap, B-Cap and C-cap. As the ICA method is heavily reliant on everything to work as planned, alternative methods were tried. Furthermore it must be noted that the overhangs of 4 base pairs is a small overhang and are seldom fit to ligate at room temperature. Redesigning the gene towards a larger overhang might create easier ligations. During ligation, constant turbation of the fluids must be applied as the beads will subside in the bottom of the tube without, creating unfavorable conditions for ligations.

Alternative method

As positive results were hard to come by (4 out of 21 tries), alternative methods were used. We tried ligating constructs which did not have overlapping overhangs in series at 16 degrees celsius overnight, as the lower temperature would increase the possibility of ligation between small overhangs. However also decrease the activity of T7 ligase, thus we added 0,2 µL of 40 mM ATP to the sample at each ligation step. To achieve a MaSp1/2 construct we tried making overnight ligations for Initiator-AB-BC and CA-Terminator. Likewise we tried for a construct of MaSp1-MaSp2; Initiator-AB-BC, CA-AB and BC-CA-Terminator. We tried these on biotinylated streptavidin beads and without, hoping the PCR would show a clear band at the desired basepairs.

Missing ligation between Thuricin S and MaSp silk monomer

Under our experiments we noticed that using the standard primers from iGEM respectively VF2 and VR for our pPCR on plasmids containing the bacteriocin with silk-overhangs would after digestion would lead to 2 bands only differing by 1 bp from each other. We thus decided to amplify our bacteriocin with silk-overhangs. However after changing primers we experienced that the pPCR gave unexplainable results including a lot of smear. Various PCR programs were tested but we never ended up with a product that could be purified and used for ICA. As another method we also tried digestion of the pSB1C3 and the Thuricin S with silk-overhangs PCR product to perform a ligation into the pSB1C3 directly by use of restrictions enzymes. This result was verificated by gelelektrophorese. However it was not possible to digest the plasmid containing the Thuricin S with silk overhangs and a purification could not be performed. For future testing it would be interesting to cut with restrictions enzymes for longer periods, or maybe design new primers for the pPCR necessary for ICA method.

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. By staining the cell cultures with nile red, we can create a relative quantitative measure for how much plastic is inside each cell. The following results are made with 4 biologic replicates of each strain. The strains were incubated for 28 hours before the measurements were made.

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

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

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

Figur tekst [Figure x: On y-axis we display the number of events and on the x-axis the corresponding intensities. Cells of the different strains are displayed in different colors. Each event represents a cell and the intensity represents the amount of PHB in that cell.]

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 these constructs are displayed in green in figure x (above). However, as seen on the figure above, the events with the largest intensities on the figure above, are from the strain containing our construct, K2018096, indicating that this construct generates more plastic, than any other hybrid promoter phaCAB in iGEM. 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: “s” = strong, “W” = weak and “MS”=medium. The y-axis displays 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 of our flow cytometry analysis strongly suggest thatthe strongest additional promoter does not produce the largest amount of plastic. Without further qualitative data, we can only speculate as to why this is the case.

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

The figure below illustrates our thesis for the observations stated above. It is, however, important to keep in mind that we do not have qualitative data to back the thesis.

!! OBS figur mangler - hvilken skal indsættes?

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

As the above figure states, 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.&nbsp.