Overview

Working with bacteriocins

• Verification of stepwise experiments: pPCR, cPCR and Fast Digest by Gel electroforesis
• IMPACT Method
• Bacteriocin extraction
• Determining bacteriocin concentration and effect
• Performning Minimum Inhibitory Concentration (MIC) test

Creating Silk monomers and Hybrid-Silk

• Iterative Capped Assembly (ICA) method
• Assembling Silk Gene fragments

PHB production and extraction

• Digestion with Hypochlorite
• Solvent extraction
• Evaluation of extraction methods
• Developing a secretion system
• Work with S. aureus Pantothenate Kinase II
• iTRAQ analysis
• Flow cytometry
• Filament making - and 3D printing of PHB

Protocols

SOP's

Part reg.	Name	lenght (bp)	Host organism	Target Organism
K2018010	Laterosporulin	352	Brevibacillus	Staphylococcus aureus & Pseudonomas aeruginosa
K2018011	ThuricinS	259	Bacillus thuringiensis	Pseudonomas aeruginosa, Enterobacter Cloacae
K2018012	LacticinQ	361	Lactococcus lactis QU5	Staphylococcus aureus
K2018014	Laterusporulin-ThuricinS	415	Brevibacillus, Bacillus thuringiensis	-
K2018015	LacticinQ-LacticinZ	529	Lactococcus lactis QU5, Lactococcus lactis	-
K2018019	PyocinS5	1696	Pseudonomas aeruginosa	Staphylococcus aureus & Pseudonomas aeruginosa

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 contain 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.

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 contain 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 prevent the repression of the β-galactosidase coding gene LacZ in ER2566 and thus induce 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 contain 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 prevent 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..

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)

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 Demonstrate and 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 degree celsius, 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 were 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 Demonstrate and 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 1 sticky-end, that eliminates future ligations. Correct ligations will be controlled by the use of cap-sequences, that bind to the previous step 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 contains a standard prefix and the terminator contains a standard suffix. These sites makes 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 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 too 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