Difference between revisions of "Part:BBa K2918054"

Revision as of 23:50, 19 October 2019

P_BHR sp1 promoter

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

Sequence and Features

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 . 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 Transcriptional Activator like Effector protein (TALE). 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, 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 (Segall-Shapiro et al., 2018).

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

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

Characterization

Figure 1: The functioning of this part has been confirmed qualitatively in E. coli (L1U)

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

The strength of the P_BHR promoter 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 flourescence readout from Juniper GFP by flow cytometry and E.coli BL21 DE(3) 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 1 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 1. Cells of similar forward and side scatter were compared.

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

From figure 2 the median fluorescence intensity of the two samples was obatined and corrected by the flourescence of E.coli BL21 DE(3) cells. Figure 3 depicts the flourescence of GFP expression controlled by P_BHR promoter and T7 promoter.

Figure 3 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_{Bhrsp1 v2} contains the binding site for TALEsp1. The presence of the binding site seems to reduce the strength of the promoter.

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.
• 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