IMPROVEMENT OF AN EXISTING PART

Our project revolves around increasing the binding affinity of the transcription factor BlcR to the blc operator. This protein was previously used and described by the Bielefeld-CeBiTec iGEM team of 2015, after which we adapted it for our purposes by creating an E. coli codon optimized variant.

Through our Dry Lab modeling we found that the relatively low binding affinity of wildtype BlcR to DNA would cause it to dissociate from DNA even at the low levels of GHB naturally found in certain alcoholic drinks like wine. Therefore, we decided to create mutations in the DNA-binding Helix-turn-helix (HTH) domain of BlcR to create mutants with differing binding affinities to DNA. Following the procedure described in (Aditham et al., 2021), many different substitutions were created in the HTH domain. On PFAM a Hidden Markov Map from BlcR’s family was found consiting of the most occurring amino acids (AA) in each position of the HTH domain. Amino acids were changed into either alanine, valine, or a different AA from other proteins in BlcR’s family and the new mutants were designed in Benchling. Corresponding back-to-back primers were then designed with SnapGene to be able to create these mutants in vivo using site-directed mutagenesis, see module 2. Sequences containing the intended mutation were each assigned a BioBrick Part number, see the 'BlcR mutants' section of our Parts page and the corresponding Parts Registry pages.

For further 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 table below.