Team:UNSW Australia/Engineering
Engineering Success
Our team’s approach to this project was heavily influenced by the engineering design cycle. This can be seen on a macro level in our overall project structure, and on a micro level throughout the iterative development process.
As Protecc Coral’s Phase 2 Team (building on the work of the UNSW 2020 iGEM team conducted in lockdown), we had a strong design foundation before we began. This clear direction helped us to frame some goals and kickstarted the cycle, but was by no means the end of the process! In particular, the experiments conducted by Dry Lab and Wet Lab were designed to inform each other -- so that the results from one team, combined with feedback from experts and stakeholders, would naturally provide valuable information for the design and refinement of experiments conducted by the other, for an efficient, cohesive and successful project!
Heat Shock Proteins
The purpose of the small heat shock protein (HSP) system is to prevent irreversible denaturation of proteins caused by increased thermal stress. Our target heat shock protein, HSP22E, is naturally expressed in the microalgal species Chlamydomonas reinhardtii and is known to bind to denatured proteins before aggregation, and assists protein refolding with its holdase activity (Haslbeck et al, 2005).
Design
Last year, the 2020 Phase I PROTECC Coral team designed and assembled a plasmid to introduce the HSP22E gene into E. coli using a pET-19b plasmid. Their experiments demonstrated a successful expression of HSP22E in E.coli. During Phase II this year we aimed to design several assays to assess the effect of HSP22E expression on thermotolerance of E. coli cells.
We hypothesised that E. coli expressing HSP22E would have higher growth rates at elevated temperatures compared to control samples, and developed methodologies to measure growth and survival under these conditions. The subsequent design of these two testing conditions was sorted into the form of a growth and survival assay. The growth assay was designed to assess HSP22E’s ability to provide stabilisation through the growth process of E.coli at elevated temperatures. The survival assay was designed to assess if presence of HSP22E increased the survival of the E.coli cells in extreme temperature conditions.
Build
The HSP22E system was built by the Phase I team and successfully established in E. coli. The Phase 1 team carried out the initial testing for successful expression of the plasmid in the cell. Our team this year, as a Phase II project, took this pre-built system and built assays to test the effect of HSP22E expression on thermotolerance in E. coli.
We built assays to measure changes in thermotolerance through research and support of our mentors. For both assays we incorporated several controls to aid in establishing a robust protocol and considered the parameters for which we should define our experiments. For both the survival and growth assays, we considered 37°C as the best suited temperature for the control, as it is the optimal temperature for E. coli growth (Doyle and Schoeni, 1984). This ensured a benchmark for comparing normal optimal growth conditions to our tested heat stress conditions.
For both assays we also needed a control to compare the HSP22E expressing E. coli. An un-modified E. coli would not be suitable, due to when a cell expresses a plasmid it is under abnormal strain, therefore an unmodified control would perform better in optimal conditions, providing a skewed comparison. Therefore, we used an E. coli that expressed mCerulean3, a fluorescent protein as our control for testing to ensure both underwent equal strain (Markwardt et al., 2011, Watkins et al., 2013).
Another key aspect built into our assays was the inducing molecule IPTG. Both the mCerulean and HSP22E used the pET19 plasmid, which uses the lac operon to induce gene expression. IPTG is a molecule that mimics allolactose and acts to remove the lac operon repressor and induce gene expression (Kroemer, n.d). In all our testing we included a sample of HSP22E and mCerulean3 without IPTG as an additional control measure to ensure we could observe the effects of heat stress without the proteins being expressed.
Once the team had successfully selected and built controls into our assay design for the system achieved by the 2020 Phase 1 team, it was time to begin preliminary testing.
Test
We tested the growth assay to quantify the growth rate of the chassis E. coli at the control temperature of 37°C and the elevated temperature, 45°C. We measured cell growth using optical density absorbance values.
The survival assay counted the number of colony-forming units (CFUs) of the samples exposed to heat shock across a temperature gradient from the control, 37°C, to 61°C. Each assay was initially carried out once.
After the analysis of the results the data revealed several problems, with the results being inconsistent with our hypothesis and expectations.
Learn
After completing the preliminary testing of both the survival and the growth assays we found no definite conclusion could be drawn from our experimental results as both test samples and control samples were able to grow and survive at the tested temperatures without any general trend. This indicated that the designs needed to be altered and more ideas needed to be considered. We started out by troubleshooting both designs from what we learned in the preliminary testing phase. From this we re-designed the assays as follows;
- The growth assay was made to incorporate a longer incubation time after inducing expression the IPTG. This was done to ensure protein expression was occurring before testing began. Additionally the test temperature was increased to 51°C, as it was found that E. coli can grow well at 45°C and there was no thermal strain on the cells (Fotadar et al., 2005).
- The survival assay had technical issues with visualising on agar plates where smearing interfered with our colony counts. We learnt to make the agar several days in advance of testing to ensure clear visualisation. Similar to the growth assay, IPTG induction time for protein expression needed to be optimised.
Having completed the test stage of the engineering cycle, we compared the results we obtained with our design specifications to determine the effectiveness of the heat shock protein in increasing thermotolerance and whether we would need to modify the system components. Another possible reason for our unexpected results might be explained by the HSP22E peptide sequence. HSP22E contains a chloroplast transit peptide sequence that allows the protein to localise to chloroplasts (Rütgers et al., 2017). After consulting Prof. Marc Wilkins and the literature, we concluded that the transit peptide of some proteins may be cleaved off (by proteases) during membrane translocation in eukaryotic plastids (Pedrajas et al., 1999). The gene for the HSP22E in our plasmid contained the transit peptide sequence postulated by (Rütgers et al., 2017). Cleaving off the transit peptide sequence may allow the protein HSP22E to function better and result in increased thermotolerance in our model E. coli. The mature peptide was indeed chosen for structural modelling in consideration of our unexpected results, and can be tested in the next engineering cycle.
Glutathione System
Design
Corals exhibit a close relationship with their endosymbiont Symbiodinium spp. Symbiodinium provides photosynthetic products to corals while receiving inorganic nutrients such as CO2, nitrogenous compounds, phosphates from corals in return (Fransolet et al., 2012). Elevated temperature leads to photoinhibition in Symbiodinium which results in the production of reactive oxygen species (ROS) (Vidal-Dupiol et al., 2009). Leakage of ROS from Symbiodinium to corals causes oxidative stress, disrupting the symbiotic relationship and results in Symbiodinium being expelled from corals, leading to coral bleaching (Nielsen et al., 2018).
Glutathione (GSH) is a ubiquitous primary antioxidant that neutralises excess ROS by donating electrons while in its reduced thiol form (Wang et. al., 2019). To combat coral bleaching at the molecular level, we propose to introduce a glutathione production and regeneration system into Symbiodinium to enhance its antioxidant capacity and to avoid its expulsion when the coral-symbiont.
We have designed an inducible system that is activated by the presence of ROS, and drives the expression of glutathione synthetase, glutathione reductase and glutathione peroxidase for glutathione synthesis, neutralisation of ROS, and recycling of glutathione disulfide (GSSG) into its reduced form (GSH).
We chose a bifunctional glutathione synthetase (gshF) from Streptococcus thermophilus (Part:BBa_K2571001) as part of our design to catalyse a two-step reaction in the production of glutathione (GSH) (Li et al., 2011), hence simplifying our system. The gshF will be responsible for converting substrates glutamate and cysteine into γ-L-glutamyl-L-cysteine which reacts with glycine to produce glutathione. In addition, gshF is not susceptible to product inhibition, which allows a higher level of GSH to be produced (Li et al., 2011). We chose to express glutathione peroxidase (GPX5) (Part:BBa_K3750001) and glutathione reductase (GSHR1) (Part:BBa_K3750000) from Chlamydomonas reinhardtii (C. reinhardtii), which are genes that encode for enzymes that are localised in the cytosol (as opposed to mitochondria or chloroplast) (Dayer et al., 2008, Merchant et al., 2007). The two enzymes, GPX5 and GSHR1 will be responsible for recycling GSH from GSSG by catalysing the reaction of glutathione with ROS. The design idea is for our glutathione system to be present in the cytosol of Symbiodinium to neutralise ROS produced from both mitochondria and chloroplast, preventing ROS leakage into the coral. Among the five glutathione peroxidases present in C. reinhardtii, GPX4 and GPX5 were predicted to be located in the cytosol based on their N-terminal protein sequence (Dayer et al., 2008). GPX5 was chosen over GPX4 as it is more well researched and its location in the cytosol is supported by experimental data (Miao et al., 2019).
We will initially demonstrate the expression and activity of the engineered biosynthetic pathway in an E. coli system. Two plasmids will be constructed that will regulate and drive the expression of the glutathione system, which will be based on the pET-19b vector (pBR322 origin of replication, with ampicillin resistance) and pRSF-1 vector ( RSF1030 origin of replication, with kanamycin resistance).
Two plasmids using the pET-19b vector will be introduced into our chosen chassis - E. coli BL21(DE3) for the expression of our glutathione system. The first plasmid will include a constitutive promoter (Part:BBa_J23102) and a ribosome-binding site (Part:BBa_B0034) to express ROS-inducible transcription factor OxyR (Part:BBa_K1104200). The second plasmid contains ROS-inducible promoter TrxCp (Part:BBa_K1104201) for glutathione synthetase, peroxidase and reductase respectively. Once OxyR is activated in the presence of ROS, it will bind to TrxCp and guide RNA polymerase to transcribe the glutathione enzymes.
The second plasmid (pET-19b with ampicillin resistance) will be engineered to simultaneously express the three enzymes (glutathione synthetase, glutathione reductase, glutathione peroxidase). Since the gene order in a plasmid will affect the level of protein expression for each individual enzyme (Mädje et al., 2012), the wet lab team has consulted the dry lab team to investigate the best ordering of enzymes for optimal expression.
To inform our plasmid design, the dry lab team conducted analysis of available kinetic values for the three enzymes and constructed a kinetic model to study the system’s behaviour. After some extensive research, we came to the conclusion that the order of the three glutathione enzymes should be GPX5 → gshF → GSHR1, as GPX5 was found to be the least active and GSHR1 was speculated to have the highest enzymatic activity.
To test the function of our design, survival assays would be conducted to assess whether the introduced glutathione system enhances the antioxidant capacity of the organism at elevated ROS levels. We hypothesised that E. coli expressing glutathione synthetase, reductase and peroxidase would have increased glutathione production and would demonstrate enhanced survival at elevated ROS level compared to control.
Build
The two plasmids will be assembled by cloning in synthesised DNA that encodes for transcription factor OxyR, promoter TrxCp, bifunctional glutathione synthetase (gshF), glutathione reductase (GSHR1) and glutathione peroxidase (GPX5) using Gibson assembly approaches (New England Biolabs Inc.). Assembled plasmids will be transformed into E. coli, and colonies selected and grown as cultures, followed by plasmid extraction and sequencing to confirm fidelity of the constructs. Subsequently, the two plasmids will be co-transformed into E. coli and colonies will be selected through resistance towards both ampicillin and kanamycin.
Test
The ability of our glutathione system to respond to ROS and have antioxidative effects will be tested through a series of experiments. We will first conduct a survival assay to investigate whether enhanced antioxidant capacity would demonstrate enhanced survival at elevated H2O2 levels. Next, to verify if our system is functioning as intended, we will conduct a GSH-Glo™ Glutathione assay to detect and quantify the concentration of glutathione (GSH) produced. We will also perform Bugbuster protein extraction followed by SDS-PAGE to assess the level of expression for the three glutathione enzymes. In addition, we will conduct experiments to measure kinetic parameters - KMand Vmax of the three glutathione enzymes to facilitate dry lab with the modelling of the glutathione enzymes.
Learn
If our proposed experimental design of the glutathione system did not produce expected results, we may consider the following to improve our design and build.
Initially, we had planned to express the glutathione enzymes in an E. coli chassis. However, since Symbiodinium goreaui is a eukaryotic microalgae, glutathione activity would need to be characterised in a eukaryotic model (such as yeast) following experimentation with E. coli. This will be done through using appropriate yeast protein expression vectors, transcription factors and promoters. The Saccharomyces cerevisiae vector will consist of a Yap1 transcription factor and CCP1 promoter. The CCP1 promoter consists of binding sites for Yap1 and Skn7 which are transcription factors activated by oxidative stress (Part:BBa_K1907005). Similar to our E. coli chassis, the glutathione system should be expressed when the organism is subjected to high ROS concentration levels.
In addition, we must also consider how homeostasis between glutathione and ROS is maintained in the organism. As an example, ROS is essential for higher order mammals to produce immune responses (Rutault et al.,1999), while glutathione is necessary to remove excess ROS for antioxidative and ageing prevention (Sen and Packer, 2000). Homeostasis of ROS and glutathione levels is essential to keep cellular functions in balance.
Similarly in microalgae, homeostasis of ROS and glutathione may be necessary for similar functions and overall cellular health. In humans, nrf2 is a transcription factor involved in the glutathione pathway by regulating the biosynthesis of glutathione enzymes (Harvey et al., 2009). We should investigate the presence of nrf2 or equivalent in Symbiodinium to understand the mechanism and its effects when glutathione is overexpressed.
Moreover, Thioredoxin (Trx) is another major antioxidant for regulating cellular redox homeostasis (Prast-Nielson et al., 2011). Upon further investigation, we found that GPX5 is a non-selenocysteine, thioredoxin-dependent glutathione peroxidase (Fischer et al., 2009). It contains a cysteine residue site where Trx interacts with the enzyme (Dayer et al., 2008). We hypothesised that the Trx content in our experimental chassis should suffice for the function of GPX5. However, if our system does not function as intended, we would conduct further characterisation of GPX5 function and consider the possibilities of including Trx in our experimental design. Although there is limited published research on the activity of GPX5 and its relationship with Trx, there is some evidence of Trx reducing GPX5. Hence experimental data from wet lab is necessary to determine whether our designed glutathione system is functioning as expected.
References
DAYER, R., FISCHER, B. B., EGGEN, R. I. & LEMAIRE, S. D. 2008. The peroxiredoxin and glutathione peroxidase families in Chlamydomonas reinhardtii. Genetics, 179, 41-57.
DOYLE, M. P. & SCHOENI, J. L. 1984. Survival and growth characteristics of Escherichia coli associated with hemorrhagic colitis. Applied and Environmental Microbiology, 48, 855-856.
FISCHER, B. B., DAYER, R., SCHWARZENBACH, Y., LEMAIRE, S. D., BEHRA, R., LIEDTKE, A. & EGGEN, R. I. L. 2009. Function and regulation of the glutathione peroxidase homologous gene GPXH/GPX5 in Chlamydomonas reinhardtii. Plant Molecular Biology, 71, 569-583.
FRANSOLET, D., ROBERTY, S. & PLUMIER, J.-C. 2012. Establishment of endosymbiosis: the case of cnidarians and Symbiodinium. Journal of Experimental Marine Biology and Ecology, 420, 1-7.
HARVEY, C. J., THIMMULAPPA, R. K., SINGH, A., BLAKE, D. J., LING, G., WAKABAYASHI, N., FUJII, J., MYERS, A. & BISWAL, S. 2009. Nrf2-regulated glutathione recycling independent of biosynthesis is critical for cell survival during oxidative stress. Free Radic Biol Med, 46, 443-53.
HASLBECK, M., FRANZMANN, T., WEINFURTNER, D. AND BUCHNER, J. (2005) 'Some like it hot: the structure and function of small heat-shock proteins', Nature Structural & Molecular Biology, 12(10), pp. 842-846.
KROMER, T. How Does IPTG Induction Work? [Online]. Gold Biotechnology Available: https://www.goldbio.com/articles/article/how-does-iptg-induction-work#_Toc56514562 [Accessed 22/07 2021].
LI, W., LI, Z., YANG, J. & YE, Q. 2011. Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli. Journal of biotechnology, 154, 261-268.
MÄDJE, K., SCHMÖLZER, K., NIDETZKY, B. & KRATZER, R. 2012. Host cell and expression engineering for development of an E. coli ketoreductase catalyst: enhancement of formate dehydrogenase activity for regeneration of NADH. Microbial cell factories, 11, 1-8.
MARKWARDT, M. L., KREMERS, G. J., KRAFT, C. A., RAY, K., CRANFILL, P.J., WILSON, K. A., DAY, R. N., WACHTER, R. M., DAVIDSON, M. W. & RIZZO, M. A. 2011. An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching. PLoS One, 6, e17896.
MERCHANT, S. S., PROCHNIK, S. E., VALLON, O., HARRIS, E. H., KARPOWICZ, S. J., WITMAN, G. B., TERRY, A., SALAMOV, A., FRITZ-LAYLIN, L. K. & MARÉCHAL-DROUARD, L. 2007. The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science, 318, 245-250.
MIAO, R., MA, X., DENG, X. & HUANG, K. 2019. High level of reactive oxygen species inhibits triacylglycerols accumulation in Chlamydomonas reinhardtii. Algal Research, 38, 101400.
MILLIPORE (2008) BugBuster Extraction Reagent. Available at: https://www.merckmillipore.com/AU/en/product/BugBuster-Master-Mix,EMD_BIO-71456?bd=1&fbclid=IwAR2R7M-_6s_rzlckiW0sYKwYnyVpUBUm-lhbaBrXmCpZ05z4g_7pVj_rJcs#anchor_USP (Accessed: 17 June 2021).
NIELSEN, D. A., PETROU, K. & GATES, R. D. 2018. Coral bleaching from a single cell perspective. The ISME Journal, 12, 1558-1567.
PEDRAJAS, J. R., KOSMIDOU, E., MIRANDA-VIZUETE, A., GUSTAFSSON, J. A., WRIGHT, A. P. AND SPYROU, G. (1999) 'Identification and functional characterization of a novel mitochondrial thioredoxin system in Saccharomyces cerevisiae', J Biol Chem, 274(10), pp. 6366-73.
PRAST-NIELSEN, S., HUANG, H.-H. & WILLIAMS, D. L. 2011. Thioredoxin glutathione reductase: Its role in redox biology and potential as a target for drugs against neglected diseases. Biochimica et Biophysica Acta (BBA) - General Subjects, 1810, 1262-1271.
RUTAULT, K., ALDERMAN, C., CHAIN, B. M. & KATZ, D. R. 1999. Reactive oxygen species activate human peripheral blood dendritic cells. Free Radical Biology and Medicine, 26, 232-238.
RÜTGERS, M., MURANAKA, L. S., MÜHLHAUS, T., SOMMER, F., THOMS, S., SCHURIG, J., WILLMUND, F., SCHULZ-RAFFELT, M. AND SCHRODA, M. (2017) 'Substrates of the chloroplast small heat shock proteins 22E/F point to thermolability as a regulative switch for heat acclimation in Chlamydomonas reinhardtii', Plant Mol Biol, 95(6), pp. 579-591.
SEN, C. K. & PACKER, L. 2000. Thiol homeostasis and supplements in physical exercise. The American Journal of Clinical Nutrition, 72, 653S-669S.
VIDAL-DUPIOL, J., ADJEROUD, M., ROGER, E., FOURE, L., DUVAL, D., MONE, Y., FERRIER-PAGES, C., TAMBUTTE, E., TAMBUTTE, S. & ZOCCOLA, D. 2009. Coral bleaching under thermal stress: putative involvement of host/symbiont recognition mechanisms. BMC physiology, 9, 1-16.
WANG, Y., LI, H., LI, T., DU, X., ZHANG, X., GUO, T. & KONG, J. 2019. Glutathione biosynthesis is essential for antioxidant and anti-inflammatory effects of Streptococcus thermophilus. International Dairy Journal, 89, 31-36.
WATKINS, J. L., KIM, H., MARKWARDT, M. L., CHEN, L., FROMME, R., RIZZO, M. A. & WACHTER, R. M. 2013. The 1.6 A resolution structure of a FRET-optimized Cerulean fluorescent protein. Acta Crystallogr D Biol Crystallogr, 69, 767-73.
WEBSTER, J. M., DARLING, A. L., UVERSKY, V. N. AND BLAIR, L. J. (2019) 'Small Heat Shock Proteins, Big Impact on Protein Aggregation in Neurodegenerative Disease', Frontiers in pharmacology, 10, pp. 1047-1047.