PARTS

Favorite parts

♥ Favorite Basic Part: BBa_K4361021, the BlcR-binding DNA oligo designed in collaboration with the DTU iGEM 2022 team. By analyzing the family of blc operators found in different strains of Agrobacterium tumefaciens, a consensus sequence was found. It was experimentally shown that this sequence was able to better bind BlcR than the original wildtype sequence. Additionally, the analysis showed highly conserved regions in the sequence which have not been previously described in literature, and could suggest a yet unknown binding mechanism of BlcR to DNA (Figure 1).

♥ Favorite Composite Part: BBa_K4361103, E. coli codon optimized BlcR with 6xHis-tag and TEV protease cleavage site (see Figure 2). BlcR is the main focus of our project, with our variant forming the basis not only of the part improvement aspect of SPYKE, but also being crucial to experiments performed in all of our modules. Each aspect of this composite part was essential in ensuring that our experiments could continue. The codon optimization allowed for increased expression of our protein, after which it could be efficiently purified with the His-tag. Subsequent cleavage at the TEV protease site resulted in a pure, fully functional protein. Future teams may take BBa_K4361103 as an example for the optimization of their own protein expression, benefitting from the increased efficiency of their full experimentation workflow.

For detailed information on the biology, design, and experimental results of each Part, please be referred to the individual BioBrick pages on the Parts Registry by clicking the Part name in the tables below.

blc operator DNA oligos

BlcR binds specific DNA sequences in vivo containing two sets of inverted repeat pairs, each consisting of a sequence followed by its reverse complement. One of our approaches in tackling the problem of improving the binding affinity between DNA and BlcR was to design new sequences to which BlcR could bind more strongly than the original. This resulted in 19 small dsDNA molecules containing different arrangements and variants of the BlcR-binding sequence originally found in A. tumefaciens.

Synthesis

To be able to perform experiments with BlcR, we first had to express and purify it. These 15 BioBricks were used by us throughout the project in order to produce the protein in high concentrations. By attaching a tag to BlcR, we were able to use different methods of purification to reduce contamination to an absolute minimum and obtain pure, functional BlcR for our other experiments.

Site-directed mutagenesis primers

Our second approach to improving the binding strength of BlcR to DNA was to rationally design mutants of the protein, each containing a single nucleotide substitution in the DNA-binding domain. To be able to create these mutants in vitro, we back-to-back primers for each mutation. From this library, 28 were proven to have created successful mutants and were documented as Parts.

BlcR mutants

After site-directed mutagenesis of BlcR and sequencing of the resulting mutants, 20 successful mutants were confirmed to have been created. The mutants were then tested for activity and documented as Parts.