In October, the team was able to go into the lab for a couple weeks to start cloning. Despite this, the steps that were done were preliminary in the cloning process. Figure 1 shows our basic experimental design steps. Therefore we would like to theorize how we may work with our wetlab project and mitigate our experimental flow through. The following information will highlight our basic experimental stages, how these experiments are designed, how they are assessed, and then finally the steps therein.
Figure 1: The basic experimental flow through of our project. Green represents the stage at which the team is at. Grey represents the other potential work steps that have not yet been conducted. Made with BioRender.com
Currently, we have been able to synthesize our initial pectinase genes into the pUC57-kan plasmid from IDT (also known as pUCidt), removing the need for blunt end cloning using the pJET 1.2. Cloning kit. This allows us to gain large amounts of insert DNA for use in ligation reactions to introduce the genes into either pET28a and pSB1C3. Traditionally, we verify our subcloning results by PCR, colony PCR or digest tests. These three avenues are used based on reagent availability and volume of sample numbers. After initial screening the final verification is done via sanger sequencing. As an alternative strategy we hope to also clone the inserts into pET28a, another effective expression plasmid. Cloning at the same time will ensure the highest chances of plasmid we can work with. As pET28a is also a RCF10 standard plasmid, other iGEM teams can use it too!
Cloning can be difficult as several unexpected results can occur such as unexpected gaps or nucleotide mutations seen in sanger sequencing during subcloning or unexpected bands in PCR samples or digestions. Beyond troubleshooting enzymes, DNA samples, buffers and the DNA sequence itself, it becomes a game of optimization and identifying where the bottlenecks may be.
Protein expression and Purification
Our basic experimental strategy is to first purify the pectinases using nickel affinity chromatography using the standard hexa-histidine tag that has been placed on the C-terminus of all the constructs. Then, the proteins would be futher purified using size exclusion chromatography. As a control, we have designed a set of the pnl, PelB, and PelC genes with a fusion partner GFP. This can be used as a visual indicator for proper expression and purification.
Although nickel affinity chromatography is an effective method to purify proteins, issues surrounding purification using this tag can be mediated by replacing it with a different affinity tag. Issues may include problems in the histidine tag being unable to effectively bind to the column and large contamination issues. The team has access to knowledge and equipment for purification via glutathione-S-Transferas (GST) or maltose binding protein tags. As well, the highlighted paper in our study by Keggi and Doran-Peterson  have an established purification method using a polyhistidine-rubredoxin affinity tag. Another potential strategy is to change the affinity purification step with a different step. As most of our pectinase proteins are fairly thermostable, a heat based purification method may be viable .
Depending on ease of expression, testing induced expression can be done with different concentrations of IPTG. For example, difficult templates can be transcribed more effectively by using less IPTG than typical procedures. Furthermore growing the cells at cooler temperatures could improve expression as well as protein folding if that were to be an issue with our proteins. Again proposing to use two different expression plasmids can also improve the synthesis of our protein. Having two different vector options for overexpression can prevent time loss if the protein is unable to be expressed in the pSB1C3 backbone.
Other issues such as protein aggregation and folding issues may occur as well. This can be troubleshooted by looking at different buffer systems to use during purification and storage or studying optimal storage conditions.
We plan to validate our protein variants’ activity using a colorimetric assay which will be done in vitro as well as with cell lysates. The colorimetric assay is very simple, in which the Pectin substrates are dissolved in a buffer, then the lyase enzyme of choice is added. After a 5 minute incubation the absorbance at 232 nm can be traced overtime using a basic spectrometer. As the pectin is broken down, the absorbance at 232 nm will increase in value.
Figure 2: An arbitrary example of the colorimetric assay where the absorbance at 232 nm is tracked overtime, chowing an increase in absorbance corresponding to a decrease in pectin levels.
The assay will determine the relative enzymatic activities of our thermostable pectinase variants as compared to the WT pectinase proteins we are currently cloning. For more information on the development of thermostable variants please see our modelling page. As we want to also assess the thermostability of the protein we will track the enzymatic activity in different temperatures to assess its optimal conditions. Determining its optimal conditions and its max thermostable conditions is crucial for the success of our project. However, in case that this assay is not able to be specific enough for use, there are other ways that we can validate protein activity in a highly more sensitive way.
Another way we may be able to test the enzymatic activity of our system is through fluorescent labelling. A pectinase assay using fluorescently labelled substrates has been done previously . Instead of relying on reading the absorbance at 232 nm, a fluorescent label can be used to specifically track a decrease in fluorescence over time. As fluorescence emitted strong signals when its excited, this could also help with any issues in our cell lysate experiments, where samples are more contaminated and spectroscopy may not be strong enough to detect pectin breakdown.
Crucial controls for our validation assay are:
- Buffer blanks containing our pectin substrate(s) of choice
- Using our wild type pectinases without the GFP fusion to compare against our thermostable variants in terms of temperature stability and activity
- If possible purchase a protein standard for our activity assay
- Have pectin substrates (commercially bought) as a control if using organically derived pectin substrates
- For using the lysate system, have a empty cell control as well as a cell control with a empty vector
Further mitigations to our project
One more mitigation that can be done with our project was at the suggestion of Dr. Wade Abbott from Agriculture Canada. If our thermostable variants are not suitable for our needs we can pivot towards using already thermostable proteins and fuse them onto our pectinases . This may remove the need for extensive computational methods and the need for screening a multitude of variants using molecular dynamics and wetlab tests.
- Keggi and Doran-Peterson. (2020) The homogalacturonan deconstruction system of Paenibacillus amylolyticus 27C64 requires no extracellular pectin methyltransferase and has significant industrial potential. Applied Environmental Microbiology. 86: e02275-19
- Olichon, A., Scweizer, D., Muyldermans, S., and Marco, A. (2007) Heating as a rapid purification method for recovering correctly-folded thermotolerant VH and CHH domains. BMC Biotechnology. 7:7
- Ishii, T., Ichita, J., Matsue, H., Ono, H., Maeda. (2002) Fluorescent labelling of pectic oligosaccharides with 2-aminobenzamide and enzyme assay for pectin. Carbohydrate Research. 337: 1023-1032
- Pierre, B., Labonte, J., Xiong, T., Aoraha, E., Williams, A., Shah, V., Chau, E., Helal, K., Gray, J., and Kim, J.(2015) Molecular determinants for protein stabilization by insertional fusion to a thermophilic host protein. ChemBioChem. 16:16