1. Team CUHK

NUSGEM initially wanted to explore using RNA aptamers as a reporter system for our stress promoter (htpG1) and blue light repressible promoter. However, we faced many difficulties in constructing our aptamers reporter system and we could not get a successful construct for many months. However, we read that the CUHK iGEM team was working on aptamers. Hence our team leader, Nanda, met up with them in Hong Kong.

After an exciting discussion in Lee Woo Sing College at the Chinese University of Hong Kong, both teams saw a great collaboration opportunity between us, discovering many aspects of our projects which we could collaborate in - wetlab, modelling and hardware.

The two teams concluded our meeting by agreeing to keep in contact, and we continued to discuss intensively on our collaboration even after Nanda returned to Singapore. Eventually, we decided to collaborate on wetlab!

As the CUHK team worked on a RNA-based project, they were urgently looking for a particular strain of E. coli, BL21 Star. It is an RNase-knockout strain that will allow them to maximize RNA production. They asked whether our lab has this E. coli strain - we do! Without any hesitation, we shipped the strain over to them.

On the other hand, NUSGEM needed help on RNA aptamers constructions. Team CUHK kindly offered their expertise by redesigning our RNA aptamer constructs, as well as designing suitable primers to increase the chances of successful construction. In addition, they incorporated the RNA aptamer that they aimed to characterise, tRNALys3-iSpinach, with our stress promoter (BBa_K2819010), for us to characterise. Both teams agreed on constructing the constructs from both teams simultaneously, using the set of primers which Team CUHK has designed. They advised us to use overlap PCR in our constructions, a technique that we don’t usually use in our lab. We took their advice and were delighted to find out from our gel electrophoresis results that our overlap PCR was successful!

With their help, we eventually succeeded in constructing the RNA aptamer plasmids! We managed to successfully construct our plasmids, as well as Team CUHK’s parts. Subsequently, NUSGEM assisted CUHK in characterising their parts. We measured the fluorescence intensity given off by the RNA aptamers under different temperatures according to the assay protocol provided by them, and found that that tRNALys3-iSpinach gives off a higher fluorescence reading than tRNALys3-Spinach 2.1 (Figure 1)!

(Figure showing characterization results - waiting for modelling to plot)

Last but not least, CUHK also hoped to carry out their survey with a wider audience, so NUSGEM did the survey and spread the word of their survey to help them with gathering responses in Singapore.

2. Team Toulouse-INSA-UPS and Team ULaval

What started out as a contacting session between Toulouse and Quebec turned into a great collaboration between three groups from different parts of the world. All three teams wanted to bring to discussion how synthetic biology touches different aspects of . The three main subjects we chose to discuss were bioethics in synthetic biology, economic aspects and lastly, society’s perspective of synthetic biology.

3.Team NTU

With Team NTU just being next door in sunny Singapore, how could we not collaborate with them? Both teams first met in our SG iGEM Meetup (hyperlink), and we subsequently met up several times to finalise our collaboration plans.

NUSGEM help Team NTU to verify and characterise their dCas9 fused with adenine base editor system in E. Coli BW25141. We co-transformed 2 plasmids into the strain, ___ and ___, and characterised the constructs under 4 different inducer conditions: No inducer, IPTG, Arabinose, IPTG+Arabinose. Their system has the following proposed working mechanism: Kan*(to confirm the name), induced by IPTG, is a construct with a mutated kanamycin gene attached to SgRNA, while dCas9 (to confirm the name), induced by arabinose, is capable of editing and correcting the mutation on Kan* to restore the plasmid’s antibiotic (kanamycin) resistance.

The editor system is only supposed to work when both IPTG and Arabinose are present, as both SgRNA and dCase9 must be induced. SgRNA provides a guide sequence to direct dCas9 to target its plasmid, to perform adenine editing on Kan* (to cfm plasmid name). To demonstrate this, we plated the cells from each condition into 2 types of agar plates with different antibiotics composition, one with kanamycin+gentamycin+chloramphenicol, and the other with gentamycin+chloramphenicol.

We verified that their system works as proposed. As shown in the picture above, only the system induced by both inducers had colonies growing on the agar plate with kanamycin+gentamycin+chloramphenicol, while no colonies were observed for the other 3 induction conditions.

As our labs lack the necessary equipment to conduct RT-qPCR, we were unable to study the level of mRNA expression of our constructs. Fortunately for us, Team NTU is well-versed in this technique, having provided assistance to many teams previously. We gratefully accepted their offer to help and passed them several of our dye-synthesizing constructs for RT-qPCR analysis and characterization. The results can be seen in Figure X.

The low levels of expression helped to pinpoint the reason for the lack of success we have in producing our target compound. It prompted us to make revisions to several aspects of our project, most notably the change of host from BL21 (DE3) to BL21 Star (DE3), a RNase knockout strain which has enhanced protein expression. Moreover, we also gained further insights into the design of our plasmids and how we can improve on them.

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