New Composite Part

As our General Biological Engineering Special Prize our team wanted to choose the New Composite Part. The decision was driven upon our team’s urge to have an impact on the world around us in addition to having an impact on the synbio community. Our project MercuLess has a heavy emphasis on real-life implementation. With whole constructs that can be utilized in real-life applications as they are, our team can have a practical impact to answer the United Nations’ Sustainable Development Goals. In the creation of MercuLess, our team created a new composite part using Synechocystis as a host organism to make a sustainable way of methylmercury bioremediation (Figure 1).

We designed a composition of many new parts for the iGEM community. In all our constructs we built on a pDF backbone. As our host organism does not yet have in silico tools to optimize the Ribosomal Binding Sites (RBS) for efficient protein translation, we used different combinations of RBSs ( RBS S3, RBS S4 and RBS S5) in our composite parts to find the most optimal one (Thiel et al., 2018).

The first RBS of the construct was followed by our gene of interest – merA. One of our genes was a native Synechocystis gene and the other was a homolog from Pseudomonas Aeruginosa. In some composite parts the merA gene was replaced by a reporter gene sYFP2. This allowed us to experiment with our genes of interest individually and also gain some important data of translation efficacy.

To start the translation of our other gene of interest, merB, a second RBS was needed to the composite. These RBS were the same as the previous ones, except they had different restriction sites. Because merB is not a native gene for Synechocystis, the homologs are either from Escherichia coli or Pseudomonas Aeruginosa. The merB homolog was followed by a transcription terminator sequence. Just like for merA, we also designed composite parts where the merB gene was replaced by a reporter gene EFE (Ethylene forming enzyme).

Figure 1. In silico Golden Gate assembly of our Composite in SnapGene. This figure shows one example of our composite assembled in silico using SnapGene. From the figure you can spot the overhangs of each individual part after they have been digested by BbsI and ligated by T7 ligase. From these parts, fragment 3 and fragment 5 were our genes of interest meaning merA and merB. The pDF backbone has been cut out of the picture, but it would attached to fragment 1 and fragment 6. Fragment 1 = PA1lacO-1-RiboJ, Fragment 2 = RBS -S3/ -S4/ -S5, Fragment 3 = merA Synechocystis / Pseudomonas Aeruginosa, Fragment 4 = RBS -S3/ -S4/ -S5, Fragment 5 = merB Escherichia coli/ Pseudomonas Aeruginosa, Fragment 6 = Transcription terminator

Our composite parts can be seen in its entirety on our Parts page. To see how these composite parts were designed, go check our Design page.