Difference between revisions of "Part:BBa K2918000"

Revision as of 11:09, 19 October 2019

P_BHR

Broad host range promoter demonstarted to work in E.coli and P. putida and expected to work in B. subtilis

Sequence and Features

Usage and Biology

The broad host range promoter 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 Pmin promoter broad host range, the 5′-TTGAAA-3′ sequence in the UAS region of the Pmin promoter was changed to 5′-TTGACA-3′ and the 5′-TTAAT-3′ in the AT rich region was changed to 5′-TATAAT-3′.

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. 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

The P_Bhr was characterized by comparing it to the T7 promoter. As a reporter, a GFP fluorescence readout was used. In order to measure fluorescence, we use a flow cytometer.

The protocol for preparation of samples for the flow cytometry assay is as follow:

1. Grow samples overnight
3. Dilute overnight cultures to OD = 0.01 into 1 mL, and grow for 2 hours on 37 degrees 250 rpm shaking in 2 mL Eppendorf tubes.
5. Dilute 1:100 into 5 mL, and grow for 4 hours on 37 degrees 250 rpm shaking in 50 mL eppendorf tubes. Induce with IPTG where necessary
7. Keep samples at 4 degrees for 1 hour