Part:BBa_K2918008

Transcription Activator like Effector protein (TALEsp1)

TALE protein with target binding site: TTATAACTCAACCTATAG.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 235
  Illegal PstI site found at 2490
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 235
  Illegal PstI site found at 2490
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 202
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 235
  Illegal PstI site found at 2490
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 235
  Illegal PstI site found at 2490
  Illegal AgeI site found at 1229
  
• 1000
  COMPATIBLE WITH RFC[1000]

This part has been confirmed by sequencing and has no mutations.

Usage and Biology

Transcription Activator Like Effectors (TALE) proteins were originally discovered in a plant pathogenic bacterial genus Xanthomonas (Doyle, Stoddard et al., 2013). These proteins consists of repeats where 12th and 13th amino acids can vary, these are called the repeat variable diresidue (RVD). RVDs have been shown to bind to DNA in a simple one-to-one binding code (Doyle, Stoddard et al., 2013):

Table 1: Overview of amino acids and nucleotide pairs

Amino Acids	Nucleotide
HD	C
NG	T
NN	G
NI	A

The direct correspondence between amino acids allows scientists to engineer these repeat regions to target any sequence they want. In our system, we used the TALE protein as a repressor by engineering promoters to contain the binding site of this specific TALE protein (0.1 T7sp1 promoter, 0.5 T7sp1 promoter and P_BHRsp1 promoter).
TALE proteins bind in a fully uncooperative manner (figure 2) (Segall-Shapiro et al., 2018) and this allowed us to implement an incoherent feedforward loop which results in perfect adaptation to copy number (figure 1).

• 

  Figure 1: Overview of incoherent feed-forward loop

• 

  Figure 2: Overview of how the TALE proteins represses GFP

Strain Construction

The DNA sequence of the part was synthesized by IDT with flanking BpiI sites and respective Modular Cloning (MoClo) compatible coding sequence overhangs. The part was then cloned in a level 0 MoClo backbone pICH41308 and the sequence was confirmed by sequencing. The cloning protocol can be found in the modular cloning section below.

Modular Cloning

Modular Cloning (MoClo) is a system which allows for efficient one pot assembly of multiple DNA fragments. The MoClo system consists of Type IIS restriction enzymes that cleave DNA 4 to 8 base pairs away from the recognition sites. Cleavage outside of the recognition site allows for customization of the overhangs generated. The MoClo system is hierarchical. First, basic parts (promoters, UTRs, CDS and terminators) are assembled in level 0 plasmids in the kit. In a single reaction, the individual parts can be assembled into vectors containing transcriptional units (level 1). Furthermore, MoClo allows for directional assembly of multiple transcriptional units. Successful assembly of constructs using MoClo can be confirmed by visual readouts (blue/white or red/white screening). Click here for the protocol.

Note: The basic parts sequences of the Sci-Phi 29 collection in the registry contain only the part sequence and therefore contain no overhangs or restriction sites. For synthesizing MoClo compatible parts, refer to table 2. The complete sequence of our parts including backbone can be found here.

Table 1: Overview of different level in MoClo

Level	Basic/Composite	Type	Enzyme
Level 0	Basic	Promoters, 5’ UTR, CDS and terminators	BpiI
Level 1	Composite	Transcriptional units	BsaI
Level 2/M/P	Composite	Multiple transcriptional units	BpiI

For synthesizing basic parts, the part of interest should be flanked by a BpiI site and its specific type overhang. These parts can then be cloned into the respective level 0 MoClo parts. For level 1, where individual transcriptional units are cloned, the overhangs come from the backbone you choose. The restriction sites for level 1 are BsaI. However, any type IIS restriction enzyme could be used.

Table 2: Type specific overhangs and backbones for MoClo. Green indicates the restriction enzyme recognition site. Blue indicates the specific overhangs for the basic parts

Basic Part	Sequence 5' End	Sequence 3' End	Level 0 backbone
Promoter	NNNN GAAGAC NN GGAG	TACT NN GTCTTC NNNN	pICH41233
5’ UTR	NNNN GAAGAC NN TACT	AATG NN GTCTTC NNNN	pICH41246
CDS	NNNN GAAGAC NN AATG	GCTT NN GTCTTC NNNN	pICH41308
Terminator	NNNN GAAGAC NN GCTT	CGCT NN GTCTTC NNNN	pICH41276

Characterization

The functioning of this TALE protein as a repressor to establish an incoherent feed-forward loop that has been extensively characterized in our project.
More information on this can be found T7 promoter based optimized iFFL system , T7 promoter based iFFL system and medium T7 promoter based iFFL system .

References

• Doyle, E. L., Hummel, A. W., Demorest, Z. L., Starker, C. G., Voytas, D. F., Bradley, P., & Bogdanove, A. J. (2013). TAL Effector Specificity for base 0 of the DNA Target Is Altered in a Complex, Effector- and Assay-Dependent Manner by Substitutions for the Tryptophan in Cryptic Repeat –1. PLoS ONE, 8(12).
• Segall-Shapiro, T. H., Sontag, E. D., & Voigt, C. A. (2018). Engineered promoters enable constant gene expression at any copy number in bacteria. Nature Biotechnology, 36(4), 352–358.