Part:BBa_K2918009

Medium T7sp1 promoter

T7 promoter variant with about 50% strength relative to wild type T7 promoter and has been engineered to contain a binding site for TALE repressor.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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

Usage and Biology

The medium T7 promoter variant was engineered to contain a binding site for a Transcriptional Activator like Effector protein (TALE). TALE proteins consist of repeats where 12th and 13th amino acids can vary, the repeats are called the repeat variable diresidue (RVD) (Segall-Shapiro et al., 2018). These RVDs have been shown to bind to DNA in a simple one-to-one binding code. A unique 18bp binding site was incorporated into the promoter, this binding site recruits a specific TALE protein called TALEsp1 that acts as a repressor (Segall-Shapiro et al., 2018). The TALEsp1 protein was designed to bind protein specifically at the binding site and are predicted not to bind anywhere in the E.coli genome. The position of the binding site can be readjusted to alter the extent of repression by the TALE protein.

Strain Construction

The DNA sequence of the part was synthesized by IDT with flanking BpiI sites and respective MoClo compatible coding sequence overhangs. The part was then cloned in a level 0 MoClo backbone pICH41233 and the sequence was confirmed by sequencing. The cloning protocol can be found in the MoClo section below.

Modular Cloning

Modular Cloning (MoClo) is a system which allows for efficient one pot assembly of multiple DNA fragments (Weber et al., 2011). 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 strength of the medium T7_sp1 promoter was characterized by comparing it to T7_{sp1 promoter}. For comparision of the promoter strengths, the two promoters were cloned in the same backbone (pICH47761 ) and were paired with the same RBS (Universal RBS). The strengths were compared by measuring flourescence readout from harmonized GFP by flow cytometry and E.coli Top10 cells were used as blank. Click here for the protocol and FCSalyzer v.0.9.18-alpha was used to analyze data from the flow cytometry experiment.

The scatter plot in figure 1 was used to gate the most dense cell regions of the blank and the same gating was considered to obtain the flourescence values depicted in figure 1.Cells of similar forward and side scatter were compared.

Figure 1: Scatter plot of forward and side scatter of E. coli Top10 cells.

Gating was performed on the data in the flourescnece histogram (figure 2) to discern between flourescent and non-flourescent cells.

Figure 2: Raw fluorescence data. The curves represent flourscenece values of E.coli Top 10 cells (black), clones with GFP expressed from T7_sp1 (red) and clones with GFP expressed from T7_sp1 (blue).

From figure 2 the median fluorescence intensity of the two samples was obatined and corrected by the flourescence of E.coli Top10 cells. Figure 3 depicts the flourescence of GFP expression controlled by T7_sp1 and medium T7_sp1.

Figure 3: Fluorescence values of medium T7sp1 and T7sp1 with median fluorescence of E. coli Top 10 cells without a plasmid substracted.

Figure 3 shows that the strength of medium T7_sp1 is approximately 64% relative to T7_sp1 promoter. We can thus conclude that the binding site for TALEsp1 does not significantly influence the strength of the promoter as the medium T7 promoter has been shown to exhibit about 50% of wild type T7 promoter (Komura et al., 2018)

References

• 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.
• SWeber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. Plos ONE, 6(2), e16765. doi: 10.1371/journal.pone.0016765
• 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.
• Komura, R., Aoki, W., Motone, K., Satomura, A., & Ueda, M. (2018). High-throughput evaluation of T7 promoter variants using biased randomization and DNA barcoding. PLOS ONE, 13(5), e0196905. doi: 10.1371/journal.pone.0196905
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