Part:BBa_K2918054

P_BHR sp1 promoter

Broad host range promoter with a binding site for Transcriptional Activator like Effector (TALE) repressor protein.

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]

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

Usage and Biology

The broad host range promoter (P_BHR) has been designed by combining promoter regions of E. coli, B. subtilis and S. cerevisiae (Yang et al., 2018). The promoter is based on the P_min minimal promoter of S. cerevisiae. It was found that the conserved -35 and -10 regions (5′-TTGACA-3′ and 5′-TATAAT-3′ respectively) were the same in E. coli and B. subtilis . Therefore, to make the P_min promoter broad host range, the 5′-TTGAAA-3′ sequence in the UAS region of the P_min promoter was changed to 5′-TTGACA-3′ and the 5′-TTAAT-3′ in the AT rich region was changed to 5′-TATAAT-3′.
The broad host range promoter (P_BHR) was engineered to contain a binding site for a TALE protein. The 17bp spacer between the -35 and -10 conserved sequences (5′-TTGACA-3′ and 5′-TATAAT-3′ respectively) was replaced with a binding site (17bp) for TALE protein. 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 17bp binding site was incorporated into the promoter, which 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 (Segall-Shapiro et al., 2018).

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  Figure 1: Design of TALEsp1-regulated version of T7 promoter variants was done by annexing the operator sequence directly downstream of the 23 base pair promoter.

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.

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

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  Figure 2: The functioning of this part has been confirmed qualitatively in P. putida

The strength of the P_BHR promoter with TALEsp1 binding site was characterized by comparing it to T7 promoter. For comparison 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 fluorescence readout from Juniper GFP by flow cytometry where E. coli BL21 (DE3) cells were used as blank. Click here for the protocol. FCSalyzer v.0.9.18-alpha was used to analyze data from the flow cytometry experiment.
The scatter plot in Figure 3 was used to gate the most dense cell regions of the blank and the same gating was considered to obtain the fluorescence values depicted in Figure 4. Cells of similar forward and side scatter were compared.

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  Figure 3: Scatter plot of forward and side scatter of E. coli BL21 (DE3) cells.

Gating was performed on the data in the fluorescence histogram (Figure 4) to discern between fluorescent and non-fluorescent cells.

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  Figure 4: Raw fluorescence data. The curves represent fluorescence values of E.coli BL21 (DE3) cells (black), clones with GFP expressed from T7promoter (pink) and clones with GFP expressed from P_BHR (blue).

From figure 4, the median fluorescence intensity of the two samples was obatined and corrected by the fluorescence of E.coli BL21 (DE3) cells. Figure 5 depicts the fluorescence of GFP expression controlled by P_BHR promoter and T7 promoter.

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  Figure 5: Fluorescence values of T7 and P_BHR with median fluorescence of E. coli BL21 (DE3) cells without a plasmid subtracted.

Figure 5 shows that the strength of P_Bhr in E. coli is significantly higher than a T7 promoter induced using 1 mM IPTG for 4 hours. P_Bhr contains the binding site for TALEsp1.

References

• Yang, S., et al. (2018). "Construction and Characterization of Broad-Spectrum Promoters for Synthetic Biology." ACS Synthetic Biology 7(1): 287-291.
• 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.
• S Weber, 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