Team:Hong Kong HKUST/Human Practices
Over the course of our project, we have asked for advice from various experienced professionals as well as given back to the synthetic biology community by participating in and holding public events to raise awareness about iGEM and synthetic biology. his page shows what we have done for:
Because our project is aimed at advancing current circuits, we expect our bistable system to be further developed and used by other synthetic biologists. Therefore, we chose to engage with the biological research community by consulting them for advice and comments.
Advice from Dr. Alan Siu Lun Wong
We approached Dr. Alan Siu Lun Wong, an assistant professor for the University of Hong Kong School of Biomedical Sciences, who’s research focus is on the utilization of CRISPR-based genome engineering to find new biomedical and biotechnological solutions for complex human diseases. Upon the explanation of our circuit, he mentioned that the circuit would theoretically work if we were to optimize the following factors: gRNA efficiency, dCas9 expression, promoter strength and stoichiometry. However, he also raised some concerns regarding our glowing plant application, that using sunlight to induce promoters is difficult and that there are no existing light-sensitive promoters.
To address this concern, we integrated chemically inducible promoters in our circuit. Another insight we gained was concerning the toxicity of dCas9 in high levels [1]. We were told that this phenomenon is rare, and is unlikely to affect our circuit design. This helped us streamline the modelling because one big part of it was to figure out the maximum strength of the constitutive promoter that can be used for expression of dCas9 before the onset of lethality. With his help, we have weeded out this unnecessary part of modelling.
Advice from Health Safety and Environment Office of HKUST
To address the biohazards of introducing genetically modified organisms into the environment, we contacted the Health Safety and Environment Office (HSEO) of HKUST. An official, Kaman Lo from the HSEO came to do an inspection of our laboratory. Upon inspection, she approved the laboratory practices conducted by the iGEM team. We also learned that E. coli (our chassis) has little risk involved as it belongs to Risk Group 1. Therefore, using them in glowing plants would not be a concern in terms of biosafety. However, there are no policies governing the implementation of such an application on campus. Hence, the safety committee must be approached to come up with a policy if we were to implement the glowing tree idea in HKUST.
Along the way of our project, we have interviewed some experts in the field. They gave us precious advice and suggestions for improving our project and experiment design. We have adopted some of these suggestions and integrated into our project.
Possible Competition between Promoters of the Same Type
We also approached Dr. Fei Sun, an assistant professor for Chemical and Biological Engineering at HKUST, to ask for his opinion on our overall project and the possibility of applying the proposed circuit as a research tool. Similar to the response from Professor Wong, he did not see glaring flaws in the circuit design in terms of logic and theory.
However, one of his postdoctoral fellows pointed out that the three constitutive promoters in our proposed circuit may display competitive behaviour among each other, thus leading to reduced protein expression by our circuit. To address his concern, we have decided to conduct an experiment assessing the level of interference between different numbers of promoters. From the result of that experiment, we are able to conclude that the competition caused by three same promoters has no significant effect on our circuit.
Aim
This experiment is designed and conducted in response to the concern expressed by Dr. Fei SUN that using multiple constitutive promoters in the same circuit may result in interference between promoters. He suggested that competition for the same transcriptional regulatory factors (TF) may result in imbalanced or reduced protein expression.
Flow of the experiment
We therefore designed experiments to assess the level of gene expression under a consecutive set of a number of constitutive promoters. The experimental constructs are pSB1C3 plasmids containing 1, 2 and 3 promoter-RBS sequences in an RFP-containing backbone, i.e. n={0,1,2}. These constructs are sequentially added using EcoR1, Xba1 and Spe1 sites.
Here are all the protocols we used for the Competition between Promoters Characterisation assay
link to our Competition between Promoters Characterisation assay protocols
DH5-α E. coli transformants of the plasmids, namely 1P (n=0), 2P (n=1), and 3P (n=2), were incubated overnight to reach saturation. The next morning, saturated cell cultures are diluted to a designated OD ready for exponential growth. Diluted cell cultures are then incubated. Every 0.5 hours over a 7.5 hours span, a portion of the cell culture is quenched. After samples are taken from every time point, i.e. after 7.5 hours, OD600 and fluorescence level (ex:586nm; em:611nm) are measured by a plate reader. The amount of fluorescent protein in a single cell is implied from the fluorescence/OD values.
Results
The graph shows no notable correlation between the number of promoters present in the plasmid and the level of fluorescence protein expression. Our model circuit only contains 3 constitutive promoters (all BBa_J23104), which is the same case as the experiment conducted. Accordingly, we conclude that there is no significant restraint in protein expression in our model circuit.
However, we suspected curves without significant correlation, as the one above, can only be replicated when the experiment is done with small numbers of promoters. Below we discuss what might be the reasons.
It is speculated that the amount of FTs is largely in excess in an E. coli cell. Therefore, the increase in the number of promoters from 1 to 3 might not be significant compared to the abundance of TFs. The constitutive promoter BBa_J23104 uses sigma factor σ70, which is the primary sigma factor in E. coli which transcribes most genes in the log phase[2]. Together with the fact that the experiment is done in the log phase, it is reasonable that the bacteria has a large intrinsic abundance of corresponding TFs, leading to insignificant competition.
Still unsure of the cause of the results, we would like to do experiments in the future using other independent variables. We will also consider the cell type, plasmid copy number, and phase when the experiment is conducted.
For human practices, our team also wanted to foster public interest for synthetic biology and the iGEM competition. We thought that the best way to do this is for us hold public engagement events. Scroll down to see what we did!
iGEM High School Camp
During early August in 2019, the Joint Secondary School iGEM camp(hosted by team Hong_Kong_JSS) was held at HKUST and our team members volunteered to be helpers. Throughout the 3-day camp, secondary school students learned about basic DNA recombinant experiments such as PCR, gene cloning, and gel electrophoresis. Students also learn about fundamental knowledge in synthetic biology. On top of lessons, our team members also held fun synthetic biology-related activities that helped spur the students’ interest in this field. The most meaningful campaign of the iGEM camp was the project brainstorming session, which encouraged the students to discuss how to use synthetic biology to achieve world-changing goals.
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HKUST Information Day Video
The Information Day at HKUST is an annual event that attracts hundreds of visitors to learn more about the university. Booths, talks, and performances are conducted throughout the campus for an entire day. To celebrate this event, we made a video that briefly introduces the HKUST iGEM team to the public. It features how HKUST first joined iGEM, previous achievements, as well as a brief introduction to iGEM competition. We hope that this video can promote public interest for the iGEM competition and add more color to the Information Day.
Watch our video here!!
SciFest a The Hong Kong Science Museum
In order to promote biology education in Hong Kong, as well as introduce synthetic biology to the general public, we wrote commentaries for the HK SciFest with the theme of STEM (Science, Technology, Engineering and Mathematics) in the exhibition on The Hong Kong Biology Olympiad & iGEM held in Hong Kong Science Museum. Fundamental concepts of synthetic biology were illustrated, including:
1. The Central Dogma
2. Polymerase Chain Reaction (PCR)
3. DNA Recombinant Technologies
Also, to give the participants an overall image of biology competitions, we explained the 2018 iGEM project PEkachu which engaged in a very creative idea - generating electricity from the metabolism of degraded polyethylene. Since our university, HKUST, is the holding site of the International Biology Olympiad - Hong Kong Contest 2019, we also tried exposing more people to the International Biology Olympiad and training program.
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To make the exhibition more interesting and readily comprehensible, we brought in many simplified graphs to illustrate professional biology knowledge. Simultaneously, we added in the pictures taken in our labs, so the viewers could get a basic idea of what biology lab work looks like. With the harmonious and hardworking atmosphere reflected in the photos, we were sure to convey the beauty in biological research to the viewers. By comparing seemingly rocket-science molecular biology concepts with daily examples, we demonstrated how synthetic biology can be related to daily life.
References:
[1] Cui, L., Vigouroux, A., Rousset, F., Varet, H., Khanna, V., & Bikard, D. (2018). A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9. Nature Communications, 9(1), 1912.
[2]J. MIKI, I. AKIRA, U. SUSUMU, and I. AKIRA, “Regulation of RNA Polymerase Sigma Subunit Synthesis in Escherichia coli: Intracellular Levels of Four Species of Sigma Subunit under Various Growth Conditions,” vol. 178, no. 18, Sep. 1996.