Team:Chalmers-Gothenburg/Design

iGEM Chalmers Gothenburg 2020

Design

Overview
Once we decided to focus on degrading Elastane, we started planning the laboratory procedure in detail. Our laboratory design is based on the chemical structure of Elastane and involves the identification and heterologous expression of enzymes to create a degradation pathway. On this page, we present the process step-by-step. This includes enzyme identification, the design of our expression system and the framework for our laboratory process. In the final section of this page, we discuss experiments and improvements of our wet lab design that we hope will help the work of future researchers.

The Elastane degradation pathway
The Elastane fibre consists of two distinct structures, as presented in figure 1. The soft segment is either polyethylene terephthalate (PET) or polyethylene glycol (PEG), depending on the manufacturer. The hard segment consists of units of polyurethane (PU). When making garments, other natural or synthetic fibres in different textiles, for instance cotton, are spun around the Elastane fibres [1]. In order to apply the enzymatic degradation of Elastane as a recycling method for textiles containing Elastane and cotton blends, the enzymes should not affect the cellulose chains that cotton consist of [2].
Elastane Molecular structure Figure 1. The chemical structure of Elastane. Elastane fibres are separated into two segments: a soft segment and a hard segment. Left of the figure shows a soft segment and this is known as PET or PEG. Right part of the figure shows a hard segment which is structured by PU.

Using the online resource Plastic Microbial Biodegradation Database (PMDB) [3], we were able to identify several enzymes that directly or indirectly use the Elastane fibre as substrate. The specific enzymatic reactions were gathered using Uniprot® and compiled into the enzymatic degradation pathway presented in Figure 2. The pathway is presented for the soft and hard segment separately.

Since the soft segment can consist of either PET or PEG, our constructs will include enzymes able to degrade both plastics. In previous competitions, several iGEM teams have focused their project on the PET degrading enzymes PETase and MHETase [4]–[6], so we chose the same strategy for degrading the soft segment when it consists of PET. Upon further research, an engineered version of PETase, ICCG, was chosen as it had shown higher levels of activity than other PETases [7]. If the soft segment consists of PEG, it could be degraded by PEG-DH, a dehydrogenase enzyme originating from Sphingomonas macrogoltabidus [8]. be

The hard segment, consisting of polyurethane monomers, calls for a more complex degradation approach. When searching for enzymes able to degrade polyurethane, we found an article providing evidence that Polyurethanase A (PueA) from Sphingomonas chlororaphis has a major role in the degradation of polyurethane into diisocyanate, ethanol, carbon dioxide and ammonia [9]. This degradation was achieved already by the imperial college iGEM team in 2013 [10]. A previous study suggested that diisocyanate can then be further transformed into diphenylmethane by a deaminase [11]. As previously shown, diphenylmethane can be further degraded through the Bph pathway [12]. This includes 5 enzymes, namely BphA1, BphA2, BphB, BphC and BphD, that are naturally expressed in Pseudomonas sp. [13]. The final products of this pathway are benzoate and 2-oxopent-4-enoate. Benzoate is likely to be accumulated, but 2-oxopent-4-enoate can be redirected to the citric acid cycle by E. coli [14].
Soft Segment
Hard Segment
Figure 2. The biological degradation pathway of Elastane. This figure shows the degradation pathway and each enzyme which contribute for Elastane degradation. The figure on the left is a pathway for the soft segment, while the one to the right shows the degradation pathway for the hard segment. The soft segment starts with PET or PEG and is degraded into EG, and the hard segment starts with polyurethane and is degraded into Benzoate and 2-oxopent-4-enolate.

Expression system

Choice of chassis

As a chassis, primarily the yeast Saccharomyces cerevisiae and different strains of Escherichia coli were considered. These microorganisms have been used as model organisms for a long time [15] [16] and are consequently very well documented, effectively facilitating troubleshooting and optimization. Even though S. cerevisiae is the most used microorganism in our lab, we decided to work with E. coli. This decision was mainly based on time constraints; E. coli grows much faster than S. cerevisiae, and we had a very limited amount of time to achieve our experimental goals in the lab. The ongoing pandemic also tied into this decision. The situation with COVID-19 meant that we could not know if our lab access would be impaired, further stressing the importance of having a time-effective chassis. Considering all of this, E. Coli was selected for our project.

To decide which specific strain of E. coli to use, the intuitive choice was the strain DH5-α, as competent cells of this strain are produced continuously by a group in our lab. Using this strain, we would save even more time that we would otherwise spend on preparing the competent cells ourselves. However, there are mutant strains of E. coli that have been evolved to use EG as carbon and energy source. One of these is the K-12 sub-strain MG1655 [17]. Therefore, we aimed to produce competent cells of this strain and using it as our final chassis, so that the bacteria would be able to survive on the products of Elastane degradation.

Plasmid backbones

When selecting the plasmid backbones, several aspects were taken into consideration. First, the two backbones had to have different resistance markers to ensure that colonies with both plasmids could be selected for. Additionally, we wanted to have markers providing resistance to some common antibiotics, so that we could avoid the cost and time of ordering exotic antibiotics. Second, considering the number of genes that would be included in each plasmid, we preferred a shorter backbone to avoid the trouble of transforming large plasmids into our chassis. Third, high copy plasmids were chosen. The copy number ties into the level of expression; a high copy number means that more DNA can be translated, which leads to greater expression of our gene. Finally, one of the most important aspects in picking our backbone was whether we had access to it or not. Having immediate access to the backbone would save us a lot of time.

After considering different candidates, we decided on using pBluescript-KS(+) and pSB1C3, two high copy plasmids that are similar in length, but differing in reporter gene as well as antibiotic resistance – pBluescript-KS(+) utilising blue/white screening with ampicillin resistance, pSB1C3 having a Red Fluorescent Protein (RFP) as a reporter gene and chloramphenicol resistance. Also, these candidates were both available in the lab.

RBS and promoter choice

The RBS and promoters used were taken from the iGEM parts registry. We chose them to obtain a high and constitutive yield of enzymes. Three different promoters from the registry were characterized (see Promoter characterization) in terms of expression levels before BBa_J23106 was chosen for all plasmids used in the project.

Gene block operon design
Since we wanted to assemble 9 different genes, cloning everything into a single backbone would be inefficient and difficult. Therefore, we made the decision to split up our pathway into two separate operons - cloning each operon into the two separate plasmids chosen. As illustrated in Figure 3, the hard segment Bph pathway enzymes were expressed from gene block A, and the soft segment degrading enzymes PETase, MHETase and PEG-DH were expressed from gene block B, as well as the first hard segment enzyme PueA.

Figure 3. Schematic illustration of the two gene block plasmids with each operon. Left plasmid shows Gene-block A which has a pBluescript-KS (+) backbone, codes for Ampicillin resistance and five Bph enzyme genes. Right plasmid is a Gene block B which has a pSB1C3 backbone, codes for Chloramphenicol resistance and other four enzyme genes. Both operons are expressed under the J23106 promoter.
The order in which the enzymes were introduced was randomly set, with the intention of changing it according to measurement and modelling results later. Genes that are expressed early in the operon tend to have higher expression, which means higher enzyme production. Thus, the order could later be changed according to the efficiency of the different enzymes.

His-tag block segment design
While gene block operons A and B were the main vector designs in the project, we soon realized that we would need to be able to express and purify the enzymes separately in order to measure their individual activity and eventual metabolic burden of the chassis. Thus, the his-tagged block segment design took shape and resulted in the plasmids presented in figure 4. pSB1C3 was used for each his-tagged block segment and the same RBS and promoter as in the gene block design.
Figure 4. An overview of the block segment plasmid. This is an example plasmid for each block segment with his-tag. The backbone is pSB1C3, which codes for Chloramphenicol resistance and a his-tag is placed downstream of the gene of interest, which is expressed under the J23106 promoter.
This design enabled the expression of one enzyme per bacterial culture, so that the metabolic burden inflicted by the transgenic enzyme can be evaluated separately (if the gene block transformants did not grow it would be very difficult to find out what caused it). Furthermore, purification of each individual enzyme is required to measure their activity, and this could be achieved with the his-tag block segment design by his-tag purification of each protein extract.

Experimental design

Plasmid assembly

The his-tags and overlapping sequences for the assembly were introduced to each gene sequence by PCR with overhanging primers. To assemble both the gene block and the block segment plasmids, Gibson assembly was the method of choice. Required overlapping sequences had already been included when ordering the genes.

Transformation and screening

After assembly, the plasmids would be transformed into DH5a by heat shock. Selection for successful transformants would then be done by plating on selective plates: Chloramphenicol plates for gene block B and the block segments, Ampicillin for gene block A. For the pSB1C3 transformants (gene block B and the block segments), colonies with a correctly assembled plasmid would be selected through digest screening. As noted in figure 3 and 4, digestion sites for SacI and XhoI are included in pSB1C3. Through purification of plasmids from the transformed cultures and digestion with named enzymes, specific lengths of DNA should be obtained. These would either confirm or reject the presence of correctly assembled plasmids.

Protein expression detection and activity measurement

Bacteria with the correct plasmids were cultivated both to exponential and stationary phase in selective media and then pelleted. Proteins were extracted through sonication of the cells. To purify the enzyme of interest, his-tag purification columns were used. A western blot was then performed to confirm the presence of the his-tagged enzyme in the purified solution. Finally, the activity was measured as described in Measurements.

Future directions
Achieving and optimizing an efficient production of the enzymes in our degradation pathway is a comprehensive task that will require time and effort by future teams as well. When the production of all enzymes is successful, many exciting further experiments could be conducted. The possible effects that the enzymes could have on the cotton fibres should be considered and evaluated, since for the implementation of the project, it is important that the quality of the natural fibres is maintained. Another consideration would be the post-weaving treatments of textile fabrics; some pre-treatment of the textiles is likely to be necessary for the enzymes to get access to the Elastane fibres.

The value of researching and further developing these enzymes is not limited to the application suggested in this project, rather the opposite. These plastic-degrading enzymes can be applied in many other waste-related projects. Thus, our work should be seen as one piece in a larger context. We encourage future teams to pursue projects that facilitates the handling of all the waste humanity has produced, and we hope to see more applications of plastic-degrading enzymes in future iGEM competitions.
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