Experiments

Bacteriocins

Chosen bacteriocins

We chose the bacteriocins according to which bacterias are most frequent detected in open wounds, such as burn wounds. Strains producing bacteriocins, which target the respective bacterias present in open wound, where chosen. The genes encoding the bacteriocins from the respective producing strains, were found and codon optimized in order to create a BioBrick part only containing the bacteriocin gene. The use of bacteriocins as new antimicrobial components are of great interest when used in local treatment. In addition it seems like resistance is rarely developed towards bacteriocins. The idea is supported by Frank Møller Arrestrup.

Verification

To verify our stepwise experiments, pPCR, cPCR and Fast Digest, we used gel electrophoresis. A successful transformation were confirmed by a colony PCR with specially designed primers for each bacteriocin. The results were confirmed according to the theoretical expected length of the bacteriocin with small overhangs (1-4 nucleotides).

Good results made happy faces and plasmid purification were made from ON cultures from the respective cPCR templates.

IMPACT Method

×

Figure 1 illustrates the removal of the bacteriocin from the pSB1C3 vector into the pTXB1 vector is schematically illustrated via SnapGene History. Figure X shows SnapGene History for the hybrid bacteriocin Laterosporulin-ThuricinS as an example.

To purify our bacteriocins, we used the IMPACT method (Intein Mediated Purification with an Affinity Chitin-binding Tag), SOP0025_v01. The IMPACT method has the ability to purify native proteins in a single chromatographic step without the use of proteases. For the IMPACT procedure the vector pTXB1 was used. The vector pTXB1 contains a mini-intein (Mxe GyrA intein) that has been modified to undergo thiol-induced cleavage at its N-terminus. At its C-terminus the intein tag contain a chitin binding domain (CBD) that allow affinity purification on a chitin column (IMPACT Kit). The target protein - in this case our bacteriocins - is fused at its C-terminus to the N-terminus of the intein tag marked by the MCS (Multiple cloning site) shown in Figure 1. The transcription of the plasmid is dependent on T7 RNA polymerase which is cloned into the LacZ gene present in the E. coli strain ER2566. With the use of a cleavage buffer that contains DTT, a thiol-agent, the vector allows protein purification of a target protein by thiol-induced cleavage at the site between the intein tag and the target protein. The results were obtained through cloning into NdeI and SapI sites which allow the target proteins to be cleaved without any residual amino acids. In order to clone the bacteriocins into the pTXB1 vector from pSB1C3, specific primers were designed to add compatible restriction sites that included the restriction sites for NdeI and SapI.

Corporation between the IMPACT plasmid pTXB1 and the E. coli strain ER2566

×

Figure 2 illustrates the effect of IPTG induction on DNA level. The effect of IPTG induction on the ER2566 genome are shown on the left and the effect on the pTXB1 are shown on the right, thus illustrating how the ER2566 genome and pTXB1 act in concert when induced with IPTG.

The E.coli strain ER2566 were used for the expression of the target gene cloned into pTXB1. ER2566 carries a copy of the T7 RNA polymerase gene inserted into the LacZ gene and the T7 RNA polymerase is thereby under the control of the lac promoter. The pTXB1 contains a T7 promoter in front of our target gene and is therefore dependent on the presence of T7 RNA polymerase. To induce protein expression in ER2566, IPTG (Isopropyl β-D-1-thiogalactopyranoside) is added. IPTG binds the lacl repressor located on the pTXB1 plasmid and alter its conformation allosterically. The event prevents the repression of the β-galactosidase coding gene LacZ in ER2566 and thus induces the transcription of T7 RNA polymerase (Figure 2). By addition of IPTG we allow E.Coli to transcribe the T7 RNA polymerase through the LacZ gene and thereby indirectly induce controlled expression of our target gene through binding of the T7 promoter.

In addition IPTG is not hydrolyzed by β-galactosidase encoded by the LacZ gene as it contains a sulfur atom in its structure and thereby diverges from allolactose which is the natural inducer of the lac operon and substrate to hydrolysis by β-galactosidase. The diverge in structure of IPTG prevents the cell from degrading the IPTG and the concentration of the inducer therefore remains constant in the experiment, which allow the expression of lac-controlled genes and thus our target gene, not to be inhibited Juers, D. H., Matthews, B. W., & Huber, R. E. (2012). LacZ β-galactosidase: Structure and function of an enzyme of historical and molecular biological importance. Protein Science : A Publication of the Protein Society, 21(12), 1792–1807..

Bacteriocin extraction

×

Figure 3 shows that we extract our bacteriocins from ER2566 by using French press and how induce thiol cleavage in order to purify the bacteriocins.

To extract our protein-Intein from the ER2566 cells after induction with IPTG, we used a French Press to destroy the bacterial cell membrane. The lysate were then poured onto prepared chitin beads. Cleavage buffer containing DTT were added, thus causing thiol-induced cleavage at the fusion between the intein and our target protein. (Figure 3)

Determining bacteriocin concentration and effect

Figure 4 shows a graph of Bradford Standard Protein assay performed with BSA. The concentration [µg/mL] of BSA are plotted on the x-axis and measured OD values are plotted on the y-axis. The equation has been made by linear regression of the plotted data.

The concentration of our bacteriocins were determined according to a Bradford Standard protein assay (Figure 4). This was addressed following SOP0028_v01. We created a Bradford protein standard curve of which the protein concentration were calculated, according to the measured absorbance (Figure 4). To view final protein concentrations go to Demonstration & Results.

Minimum Inhibitory Concentration (MIC) Test

MIC tests are essential in order to establish the bacteriocins viability as an antimicrobial compound. The test studies the in vitro susceptibility of a bacterial strain towards a compound with an antimicrobial effect. This is measured by the optical density of an overnight culture after 28 hours incubated at 37 °C, with a specific concentration of a bacteriostatic and/or bactericidal compound. This would give us the data that concludes whether our bacteriocins inhibits the growth of the strains. We wanted to perform a MIC test towards P. aeruginosa and MRSA strains. This was inspired from Hans Jørn Kolmos (professor in clinical microbiology), which suggest that bacteriocins could be potent to treat MRSA carriers. In collaboration with the iGEM Stockholm team 2016 , MIC tests were performed in order to compare the effect of our bacteriocins. The iGEM Stockholm team 2016 performed a MIC test on cell lysates whereas we performed a MIC on our purified proteins. By collaborating with the iGEM Stockholm team 2016 we thus got an indication of which of our bacteriocins elicited a significant effect towards chosen target strains.

The effect of the bacteriocins were tested by MIC testing following SOP0027_v01. The MIC plates were incubated for 28 hours. To see the results of MIC test, go to Demonstration & Results.

Summary

Creating Silk monomers and Hybrid-Silk by Iterative Capped Assembly (ICA)

The ICA method

The ICA method was first described by Adrian W. Briggs et. al. by 2012 Briggs, A. W., et al. (2012). "Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers." Nucleic Acids Res 40(15), as a solid surface-based sequential ligation, which adds DNA constructs with matching overhangs individually in series. This gives way for custom designs of genes, which can later be amplified through PCR. The method is especially effective when making repetitive DNA constructs. Traditionally DNA is added to a gene through primer overhangs in a PCR, but when working with repetitive genes, the primer could recognize multiple sites and thereby cause multiple variations of the gene. The stepwise ligation method utilizes the strong chemical binding between streptavidin and biotin. The concept is to use magnetic beads coated in streptavidin, while the initiator oligo contains a biotinylated end at the 5’. This allows the magnetic beads to be continuously isolated from the solution with a magnetic rack and the supernatant can be removed. This controls a stepwise ligation construct, which ends with a terminator sequence with one sticky-end, that eliminates future ligations. Correct ligations will be controlled by the use of cap-sequences, that bind to the previous step's non-ligated constructs and end the possibility of further ligations at the failed ligations. In the end, the gene construct will consist of an initiator sequence, a coding region and a terminator. The initiator and terminator have specific primer sites, which makes it possible for pPCR of the whole synthesized gene sequence. During our experiment the used initiator contained a standard prefix and the terminator contained a standard suffix. These sites made it possible for the gene sequence to be inserted into desired vectors.

Assembling Silk Gene fragments

Silk from Nephila clavipes consists of the genes MaSp1 and MaSp2 which individually contains repetitive gene sequences. The genes have the same restriction sites in both ends so that the overhangs are consistent throughout the construct. This step by step ligation of the individual genes (e.g. MaSp1 AB + MaSp1 BC + MaSp1 CA), allows for custom designs of the silk gene both in length and in variation (e.g. 2xMaSp1:1xMaSp2). The ICA method gives way for alternative constructs, where other genes can be incorporated in between the repetitive silk genes. This freedom of construction creates the opportunity for incorporation of bacteriocin genes into the silk construct using the ICA method. Coloplast are already aware of the great qualities of recombinant spider silk and bacteriocins, however the idea of combining the two elements are completely new to them and they see a great oppertunity in it.

Summary

PHB production and extraction

The production of PHB in E. coli is fairly cheap for a plastic product. However retrieving a somewhat pure sample of PHB from the cellular cytoplasm has proved to be an expensive task. We have tested four methods of extraction in order to determine which extraction method is optimal for large scale production of PHB, considering pricing, environmental strain, required equipment, toxicity, yield and purity.

Digestion with Hypochlorite

A method for extracting biodegradable plastic, is simply digesting the cellular components with hypochlorite, where it dissolves the cellular components, and leaves the polyester behind. The advantages of using hypochlorite as opposed to digestion with surfactants (e.g. sodium dodecyl sulfate (SDS)), is that it dissolves the membrane. Thus a single treatment can provide high purity and yield of PHB. After treatment with PHB the samples were centrifuged with 4000xG. A small side effect of hypochlorite is that it causes degradation of the PHB granules which greatly reduce the molecular weight of the molecule and thereby losing some pHB. For further details on the purification technique see SOP0033_v1, Jacquel, N., et al. (2008). "Isolation and purification of bacterial poly(3-hydroxyalkanoates)." Biochemical Engineering Journal 39(1): 15-27.,Heinrich, D., et al. (2012). "Large scale extraction of poly(3-hydroxybutyrate) from Ralstonia eutropha H16 using sodium hypochlorite." AMB Express 2(1): 59.

Solvent extraction

Another method of PHB extraction is treating lyophilized biomass with a solvent in which PHB is soluble. Removing the remaining biomass by filtration and then precipitating the PHB will provide a sample with high purity. We have tested two different methods of solvent extraction with chlorinated agents.

• Chloroform, one of our solvents, is the most toxic agent we have used for extraction but it provides purity of up to 99 % PHB Ramsay, J. A., et al. (1994). "Extraction of poly-3-hydroxybutyrate using chlorinated solvents." Biotechnology Techniques 8(8): 589-594.. For further details on the PHB chloroform extraction see SOP0034 SOP0034_v01.
• Ethyl acetate as a solvent. This method involves the least toxic substances and requires no pretreatment but fairly large amounts of chemicals needed for the extraction, which provides a purity of 97-99 % Aramvash, A., et al. (2015). "An Environmentally Friendly and Efficient Method for Extraction of PHB Biopolymer with Non-Halogenated Solvents." J Microbiol Biotechnol 25(11): 1936-1943.. For further details see SOP0033_v01.

Evaluation of extraction methods

We use Proton Nuclear Magnetic Resonance (HNMR) in order to determine the purity of PHB extracted with the different methods. HNMR is a method in which the structure of a molecule is analyzed in a magnetic spectrum. The spin of different hydrogen atoms allows us to determine the amount of- and study each distinct type of hydrogen atoms. By analyzing such a spectrum, the content of the sample can be determined. For details on how we performed HMNR analysis, see protocol HNMR - Measurement of plast purity.

Developing a secretion system

Figure 5 shows in illustration of our modified Type I secretion system. The figure illustrates how PHB are secreted due to the recognition of HlyA.

In order to avoid the high costs of plastic extraction we are utilizing a type I secretion pathway for the toxin, hemolysin, to make the bacteria secrete the plastic itself. The secretion system requires only two transport proteins, hemolysin B and hemolysin D. Together these proteins are responsible for the secretion of hemolysin in pathogenic strains of E. coli. Since PHB is not a protein compound, applying the system directly, is not possible. Instead we bio-fused a domain of the toxin hemolysin A with a protein called phasin. Phasin is a protein used in R. eutropha that binds to PHB granules in order to regulate their size. Hemolysin A is the exotoxin secreted by hemolysin B and hemolysin D. By using only a short signal domain of the protein, we can tag the bio-fused protein for secretion without making it toxic. The fused Phasin-Hemolysin protein will keep its ability to attach to the surface of PHB granulates, through non-covalent interaction and thereby target the PHB for secretion RAHMAN, A., LINTON, E., HATCH, A. D., SIMS, R. C. & MILLER, C. D. 2013. Secretion of polyhydroxybutyrate in Escherichia coli using a synthetic biological engineering approach. Journal of Biological Engineering, 7.. The event will reduce the costs of plastic extraction as well as the use of toxic and environmentally straining chemicals. In addition phasin has low binding specificity, thus the system can be used for other PHA’s (polyhydroxyalkanoates) Hanisch, J., et al. (2006). "The Ralstonia eutropha H16 phasin PhaP1 is targeted to intracellular triacylglycerol inclusions in Rhodococcus opacus PD630 and Mycobacterium smegmatis mc2155, and provides an anchor to target other proteins." Microbiology 152(Pt 11): 3271-3280.. The idea of a secretion system has already been explored by the iGEM Utah Team 2009 and have been processed by Rahman et. al 2012 RAHMAN, A., LINTON, E., HATCH, A. D., SIMS, R. C. & MILLER, C. D. 2013. Secretion of polyhydroxybutyrate in Escherichia coli using a synthetic biological engineering approach. Journal of Biological Engineering, 7., of whom the development of the secretion system were inspired from. Plastic companies are very interested in use of PHB as the material in their products, but are not doing it currently because of the price of the PHB production. Using our secretion system it would be possible to lower the price of PHB, making it more attractive for the respective companies. For details of the assembly of the secretion system, check out Protocols.

PanK

PanK is a pantothenate kinase that activates the first protein in the anabolic pathway for coenzyme A. E. coli has this protein as part of it’s core genome, but the kinase from E. coli is strongly regulated by a negative feedback mechanism. However, an increase in coenzyme A and coenzyme A thioesters will inhibit the activity of pantothenate kinase. Staphylococcus aureus requires a higher amount of coenzyme A as it lacks glutathione and thus relies on a coenzyme A based reductase system, to repair oxidative damage Leonardi, R., et al. (2005). "A pantothenate kinase from Staphylococcus aureus refractory to feedback regulation by coenzyme A." J Biol Chem 280(5): 3314-3322.. As a results the staphylococcal PanK is not feedback regulated, and we have thereby taken advantage of this by adding the specific panK to our construct. The BioBrick (K1692020) containing staphylococcal PanK was created by the iGEM Standford Brown team 2015.

iTRAQ analysis

Figure 6 shows a short overview of the steps involved in iTRAQ analysis. This includes protein extraction, digestion, labeling as well as Titanium dioxid purification, HILIC fractionation and Mass spec analysis. The figure is derived from a protocol created by the Protein Research Group, Southern University Denmark.

A protein from one organism does not necessarily recognize all its native target proteins in another organism. Additionally, it could be able to phosphorylate proteins which it did not phosphorylate before. We have therefore performed an iTRAQ analysis of staphylococcal PanK (pantothenate kinase II) in E. coli. For an iTRAQ analysis, the entire proteome of the cells are extracted and digested. The peptides from each sample are then tagged with one of four different tags that binds to the free N-terminal, present on tryptic peptides. The peptides are then mixed in an equal ratio, securing that the same amount of peptides are present from each sample. The phosphorylated peptides, that are especially interesting for the analysis of a kinase, are isolated and purified by a TiO_2 purification. Lastly the non-modified peptides and the phosphopeptides are analysed separately by mass spectrometry (Figure 6).

In the mass spectrometer the different iTRAQ tags will fragmentize differently. Therefore while analysing a single spectrum, one can compare the amounts of the proteins from the different samples, simply by comparing the intensity of the reporter ions from the iTRAQ tag. The computer can thus analyse the entire proteome of four bacterial strains at once, identifying interesting differences caused by pantothenate kinase II. For detailed procedure check out iTRAQ protocol.

Flow cytometry

In order to determine the effectiveness of our different PHB producing strains we used nile red staining combined with flow cytometry. Nile red binds to PHB and as the substance is fluorescent, it can be used as a quantitative measure for the amount of plastic present in a cell Leonardi, R., et al. (2005). "A pantothenate kinase from Staphylococcus aureus refractory to feedback regulation by coenzyme A." J Biol Chem 280(5): 3314-3322.. By combining nile red staining with flow cytometry, the average amount of plastic inside every cell from different strains can be compared and thus generate a relative measure ability of the respective strains to produce plastic. For further details check out Protocols.

Filament making - and 3D printing with PHB

The Megabot 3D printer requires filaments with a diameter of 1.75mm. In order to 3D print our produced PHB a Filastruder was used. The filastruder was assembled and tested with PLA prior to use with bacterially produced PHB.

All of our 3D printing was performed on a XX Machina. The 3D model was created as an STL file and tested with PLA filaments. PHB was ordered to compare constructs containing 100% pure PHB and constructs containing our purified plastic. Morten Østergaard Andersen suggests that PHB could also be used as the implant itself, in cases where e.g. the bone needs a scaffold until it has healed. This further support the idea of trying to 3D print PHB.

Summary

Protocols

Bacteriocins

Silk

Plastic

Standard Operational Procedures

SOP's