Template:Hong Kong HKUST/ihp

Integrated Human Practice

  • TECHNOLOGY

    For a proof-of-concept purpose, our Cre-lox system is intended to illustrate how recombinase can excise the gene of interest at a certain time we desire, starting with a simple DH10b E.coli bacteria that are easy to grow and safe to use in laboratory. We were inspired by the application of recombinases in eukaryotes where only a specific gene inside the cell was knocked out without harming the organismal cell per se.

    Having received feedbacks from people joining our organized activities in Hong Kong Science Park, where majority is parents, it surprisingly shows that our samples view GMOs to be more safe if the genetically modified gene within an organism has been knocked out before the consumption. We, therefore, directed our focus on Genetically Modified Organisms (GMOs).

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  • TOWARDS OUR APPLICATION

    The introduction of GMOs varies between countries in terms of regulation and stringency. In order to explore current GMOs in Hong Kong, we were introduced to the interview with Dr. Terence Lau, a biosafety expert from Hong Kong government. Dr. Lau shared with us the current regulation and control of GMOs. To our surprise, there is yet no measure in preventing or mitigating the risks of GMOs being accidentally released in Hong Kong. The government only emphasizes on the control of genetically modified organism in some products such as vaccine and GM papaya, but they cannot cover the examination of all GM crops. In addition, industries will take risk to benefit ratios into account before considering GMOs as their profit-based business. Throughout this interview, the issue raised has inspired us to pursue deeper into the application of Genetic Containment Strategy.

    Furthermore, we looked up some past IGEM teams’ researches and ideas on how to make their construct safe prior to the release. Besides from the consideration of kill switch as their genetic containment strategy, there is no practical implementation and standardization of the strategy.

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  • WHAT IS GENETIC CONTAINMENT STRATEGY? HOW IS IT DIFFERENT FROM BIO-CONTAINMENT?

    Genetic (Biological) Containment Strategy is a measure to safeguard recombinant DNA in microorganism such that it can only be used in lab experiment but not outside. If accidental release happens, growth of modified microbes and rDNA replication should be immediately inhibited.

    On the other hand, biocontainment is a prevention of risk of genetically engineered microorganism (GEM) release by using physical containment. Due to the demand for scaling up GEM, biocontainment may not be sufficient to contain large scale of GEM. Thus, genetic safeguards can potentially prevent the escape of microbes from proliferating. 1

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  • THE POSITIVE FEEDBACK LOOP

    Dr. Lau not only shared with us some knowledge regarding biosafety. He also raised questions concerning how we can achieve the purpose of knocking out in large population of cells.

    We have considered using feedback loop to amplify signals such that sufficient receiver cells can be knocked out at time bound. The propagation of AHL signals can be simulated by modeling the rate of diffusion from the senders to the receivers and the rate of AHL being amplified again to affect peripheral cells. You may look for our modeling execution in the ‘Modeling’ section.

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  • SOME INSIGHTS FROM THE MEETUP

    We attended a conference held by the CUHK iGEM team and a biosafety conference held by HKU iGEM team. This gave us an opportunity to gather feedbacks during the Q&A session after our project description.

    Q1: Is there a chance where random homologous recombination occur within your cell?

    Q2: Is there any enzymes that can digest your engineered plasmids instead of using these recombinases?

    Q3: How can you eliminate the plasmid after it’s recombined?

    Q4: How would you use construct in particular scenarios. For example, how will your construct help if you accidentally spill the GM bacteria into environment?

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  • ANSWERS FROM THE MEETUP

    In addition to these feedback questions, we looked more into details of our constructs where improvement can further be made to reduce the safety issues. We conducted an interview with Prof. Matthew Bennett from Rice University in Houston, who is expertise in organism’s genetic system and circuits.

    A1: It was found that even though luxR sequences do not appear in E.coli in nature, there is a homologous gene found in E.coli genome called SDiA. The luxR gene, a family of SDiA gene, shares homologous sequence such that random recombination between the plasmid and genome may occur many times, although not frequent. We address this problem by finding a chassis that has the SDiA gene being knocked out. See strain JW1901-5 2

    A2: Prof. Matthew Bennett commented that endonucleases may be more efficient in targeting multiple restriction sites and we have explored more some nucleases that could do the job of DNA degradation. One efficient method to target DNA degradation is the use of CRISPR-Cas3 device, where CRISPR nuclease specifically degrades the targeted DNA, leaving the non-targeted DNA unaffected 3 There is also a nuclease that can be transiently transcribed in vivo such as Yeast homothallic switching endonuclease (HO endo) which recognizes HO recognition sites and cleaves DNA to become linearized 4.

    A3: As our project describes a method that uses recombinase to separate between the gene of interest and the plasmid containing the origin of replication and marker gene, we use modelling to simulate a scenario to measure the dilution of plasmids and how long the recombined plasmid can be degraded over time. Nevertheless, through the end of interview, the milestone of plasmid degradation we found to be more efficient remains to be the use of endonuclease. Apart from considering endonucleases such as CRISPR-Cas3 as the new choice for further improvement in our project, our team has continued to find potential application that recombinase can be compatible with (See Application page).

    A4: The desirable design for eliminating bacterial spill is to immediately knockout the construct containing GM gene without the delay of recombinase or endonuclease expression. Thus, one question raised would be ‘When and in which scenario should time delay be used?’ if time delay is not used in the accidental release of GM bacteria. Time delay can be useful in laboratory experiments where GM bacteria should not last for so long time. This does not require modification of bacteria having genetic code dependent on some nutrients to survive nor require the activation of toxin killing GM organism in the culture. Thus, the extraction of proteins secreted into culture and/or non-secreted proteins can be proceeded safer without contamination with toxic compounds and changing strain’s genomic content not available on database to prevent the escape will be less problematic.

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