TALEs Journal

Week 1: May 1 - May 3

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Week 2: May 6 - May 10

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Week 3: May 13 - May 17

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Week 4: May 20 - May 24

We did our literature search for optimization of TALE binding affinity. There were conflicting results for the optimal truncation of the N and C terminus. On N terminus, 152 (Miller et al., 2011), 147 (Mussolini et al., 2011), 186 (Slovenia’s TALEs), and 158 or 186 (Mercer et al., 2012) residues were retained. On C terminus, 28 or 63 (Miller et al., 2011), and 76 (Slovenia’s TALEs) residues were retained. However, we did determine that residues 12 and 13 (repeat variable diresidues, RVDs) of the 34 amino acid repeats demonstrate polarity and interact more strongly with DNA at the N terminus compared to the C terminus (Meckler et al., 2013). As well, various amino acid permutations of the RVDs have varying interaction specificity and strengths.

The four TALEs designed by Slovenia 2012 iGEM team, were ordered from the registry. A primer was designed to incorporate the the target sequence of one of the Slovenia TALEs ([B]) upstream of a GFP generator. This plasmid was used as a target sequence. In addition, two other TALEs were ordered from the the authors of the Meckler et al. 2013 paper.

Week 5: May 27 - May 31

We continued our literature search for a unique and stable pathogenic E. coli marker for the TALEs to bind to. Potential pathogenic O157:H7 E. coli markers: stx1, stx2 (Yoshitomi et al., 2012), uidA (Yoshitomi et al., 2003 and Feng et al., 1994), eaeA, hly, rbfE (Fortin et al., 2000), and Z3276 (Li & Chen, 2012). However, BLAST searches determined that the use of only one 18 bp sequence is not sufficiently specific and sensitive to detect pathogenic markers. Should we continue to search for only one marker or more than one or expand the detection to any toxic shiga producing strains?

In addition to searching for a pathogenic E. coli marker, we also started working on the TALE sequence and its construction. The TALE sequence chosen has a truncation in both N- and C-terminus. The truncated N-terminus has 111 residues and the C-terminus, 42 residues (Meckler et al., 2013). The amino acid sequence was taken to obtain the nucleotide sequence optimized for E. coli. Codon optimization was performed by Integrated DNA Technologies (IDT). NEBCutter 2.0 was used to screen restriction sites for PstI, EcoRI, AgeI, NotI, XbaI, NgoMIV and SpeI, all present in the Freiburg RFC and for BsaI and Esp3I. The original TALE sequence has 11 cut sites: BsaI (3), PstI (1), EcoRI (1), Esp3I (4), AgeI (2). The restriction sites were then eliminated with silent mutations, based on a E. coli codon usage table (San Diego State University, 2012 modified from Maloy, Stewart & Taylor, 1996).

The large size of the TALE prevents simple cloning, therefore we looked to Golden Gate Assembly. Golden Gate Assembly is a subcloning strategy that uses type IIs restriction enzymes, which cut outside of their recognition sequence allowing ligation into a final product that lacks any specific recognition sites (Engler, Kandzia & Marillonnet, 2008).