Team:Rotterdam HR/Drylab

iGEM Rotterdam HR 2019

DryLab


In our project for iGEM we are going to make a construct that includes zinc fingers. We found the website www.zincfingertools.org. On this website, you can find various methods to make your zinc finger arrays and one of them was to also create your own target site. To find the right target site, we were looking for a target site that isn’t present (or present with a maximum match of 3 matches in the E.coli BL21(DE3) genome). When you found the right target site by trial and error, this website designs for you the zinc finger array. We finally have found 2 target sites that we will use to make 2 zinc finger arrays. Each zinc finger array has 6 ‘fingers’.

The HRD-Kit is a system that contains different components. The head components are aptamers, zinc fingers, split- TEV protease and the enzyme β-lactamase. The goal is to get all these components into a few plasmids. Therefore we will use a polycistron. A polycistron is a piece of DNA which can be translated into RNA and bring forth different proteins to expression on the same RNA strand. We want to insert a big part of our construct into a polycistron. This big part contains the C-TEV+GSAT-linker+Zincfingerarrays and N-TEV+GSAT-linker+Zincfingerarrays. In a polycistron only 1 promoter and 1 terminator may be present, but the amount of RBS must be the same as the number of genes you want to translate.

The first attempt was to create an open reading frame of the split TEVs, GSAT-linker and the ARRAY. The polycistron has been created so that first the C-TEV ORF will be translated. The translation will stop after the translation of the 8x-HisTag. Next what will be translated is the N-TEV, but this translation has to begin first with the 8x-HisTag and then the rest of the construct. With the help of the information of iGEM team 2013 Munich, we could determine the orientation of the split-TEV construct. The 2 ORF has to be linked and also needs to be included in an 8x-HisTag. The his-tags are important so that the proteins can be purified after induction. This has been created by using the sequence: 1105 – 1340 bp position. This is the position where the ORFs are linked and also include a new RBS. To insert N-TEV+GSAT-linker+Zincfinger array and Zincfinger array+GSAT-linker+C-TEV we had to make gBLOCKS of these components. We have ordered 2 gBLOCKS by IDT, you can see the design of the gBLOCKS in figure 1 and 2. A gBLOCK or giga block are dsDNA molecules that can vary from 125-3000 bp in length.


Figure 1: Snapgene file of gBLOCK1.


Figure 2: Snapgene file of gBLOCK2.

In our system, as we describe above, we will use zinc finger arrays. These zinc fingers are indirectly bound to aptamers which will bind to our target molecule. To be able to determine the most efficient distance needed for split-TEV to come together and form one whole protein, three ‘DNA bridges’ were designed. This is used to test different distances but also orientations of the split-TEV proteins and thus find out which distance and orientation works the most efficiently for our system. The DNA bridges are designed in SnapGene and they were ordered from Twist Bioscience. To test the ideal distance/orientation between the split-TEV proteases, we designed DNA bridges that creates 3 different distances which each 3 different orientations after being digested with certain restriction enzymes. You can get these 3 distances by using only 2 different enzymes. EcoRV is restriction enzyme A and BstEII is restriction enzyme B. To double check if the enzymes have cut the desired sites, you can put the digested fragments on a gel by performing for example gel electrophoresis or gel shift-mobility assays. We’ve put an extra 150 bp in the bridge in the middle, so you can see it on a gel when the restriction enzymes have cut in the site.



Figure 3: Snapgene file of the DNA bridge.