LIBRARIES

Site-Directed Mutagenesis Library

In module 2 we aim to increase the binding affinity between the transcription factor BlcR and its specific DNA binding sequence, the blc operator. To do this, we modified the DNA binding site on the protein using a Site-Directed-Mutagenesis strategy (SDM). To construct the SDM library, we made a rational design of amino acid substitutions in BlcR.

Making rational amino acid substitutions in BlcR

We tried to modify BlcR by making single amino acid substitutions in the DNA binding domain. First, we had to identify the DNA binding domain. After reading the literature and studying the structure in PyMOL, we found a 3-stranded winged helix-turn-helix (HTH) domain at the N-terminus (Figure 1) [1][2][3].

Transcription factors from the same IclR family use N-terminal HTH domains to bind to DNA [4] (Figure 2). This information established that the HTH was the DNA binding domain. The sequence of this domain is:

Single amino acid substitutions

we searched for our protein in the PFAM database, a database of protein families that uses hidden Markov models to create numerous sequence alignments and annotate each family's members, we discovered more than 32,000 sequences for the IlcR family's HTH domain. We had some solid foundational information to work with thanks to the publication "High-Throughput Affinity Measurements of Transcription Factor and DNA Mutations Reveal Affinity and Specificity Determinants" [5]. Single amino acids were substituted based on a Hidden Markov Map (HMM). Each amino acid was changed in Pho4's helix-loop-helix domain into the matching valine, alanine, or amino acid found in the protein's orthologs. A substantial change in binding affinity was found in 56% of the 205 genes that were highly expressed, and at least 10 of these differences improved binding affinity.

We used the HMM of the IlcR family's HTH domains found on PFAM to design rational mutations (Figure 3). We wrote a guide on how to use PFAM and PyMOL to make these rational mutations [TU Delft, see contribution section].

For each amino acid (AA) in the HTH domain, the potential mutations were first listed. For all the AA, the mutations consisted of alanine and valine and around three of the most prevalent AA among orthologs found in the HMM. We had to reduce them because the number of mutations was greater than our screening capacity. Mutations were chosen using the data from the paper described above. Firstly, we decided to skip the loop domain and the residues after the 73rd as they are the least involved in DNA binding. In light of the fact that positively charged amino acids that are close to the DNA binding site improve affinity [5], we rendered all negatively charged (D/E) amino acids positive (K/R/H), as well as all of the amino acids in the recognition helix. We considered all alterations in and around the recognition helix, which serves as the primary DNA recognition domain. Lastly, since in the paper the substitutions outside the DNA contacting domain that improved binding affinity were all A/V mutations, we only included those for our non-DNA contacting domain. The library of all the mutations can be found in Table 1 underneath.

Oligonucleotides Library

In module 3 the goal is to increase the binding affinity between the DNA strand and the transcription factor, by engineering the Blc operator sequence. A set of Blc operator variants are designed in Benchling based on the wildtype sequence originally described by Pan et al. [3].

Sequence modifications

The original sequence (designated BBa_K4361001) contains two sets of different ‘inverted repeat pairs’, short DNA sequences whose ends are complementary to their own inverse sequence, separated by a 3 nucleotide spacer. According to literature, each inverted repeat pair is able to bind a BlcR dimer, with the spacing between pairs allowing for tetramerization of the protein (Figure 4) [3].

Firstly, the wildtype sequence was scrambled using the Genscript Sequence Scramble tool [6] to create an oligo containing the same nucleotides in a randomized order. Since this oligo does not contain either of the inverted repeat pairs, binding of BlcR to the oligo would be unlikely, thus can be used as a negative control to compare other sequences. This oligo was designated BBa_K4361000.

For BBa_K4361002 through BBa_K4361013, the oligos remained largely the same as the wildtype sequence, with only the sequences at the positions of the inverted repeat pairs being changed. The original inverted repeat pairs were combined in different amounts, orders, and orientations, and a number of new pairs containing small variations were created to further diversify the oligo set. These oligos all have the same length as the wildtype sequence, 51 nucleotides.

The next group of oligos covers BBa_K4361014 to BBa_K4361019. These sequences are designed to bind a different number of BlcR proteins by varying the spacer between inverted repeat pairs and as well as increasing the number of inverted repeats. The ability of BlcR to effectively bind these DNA molecules should change significantly when compared to the oligos of the original length. A number of oligos have been designed to induce BlcR multimerization beyond tetramerization, as further described in their respective part pages .

BBa_K4361020 was designed for a BlcR-Cro repressor hybrid protein. This protein has the same GHB-binding and tetramerization domain as wild-type BlcR, but now contains the DNA-binding domain of the Cro repressor protein. Cro repressor binds two specific DNA sequences itself, which replace the inverted repeat pairs of the original oligo.

Finally, through our partnership with DTU, the team has looked into possible variants of the BlcR operator. By finding sequences originating from different strains of Agrobacterium tumefaciens, they were able to derive both a consensus sequence as well as a variant operator found in a different strain of the bacterium than our wildtype oligo. The significant differences between these sequences and our own are crucial to analyze BlcR’s DNA-binding properties in further detail.