Part:BBa_K2918014

Universal RBS

Ribosome Binding Site which is functional in both gram positive and gram negative bacteria.

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.

Overview

This ribosomal binding site has been made by the use of the RBS calculator 2.0 by (Yang et al., 2017). This RBS functions both in gram negative (E. coli) and gram positive (B. subtilis). It has been demonstrated to work in P. putida .

Strain Construction

The DNA sequence of the part was synthesized by IDT with flanking BpiI sites and respective Modular Cloning (MoClo) compatible 5'UTR overhangs. For this particular RBS, the downstream overhang 'AATG' can be modified to 'ATG' since the RBS sequence ends with adenine. This design choice will allow for seamless cloning between the 5'UTR and coding sequence leaving no part-junction sequences. The RBS was then cloned in a level 0 MoClo backbone pICH41246 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). For the protocol, you can find it here.

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

References

• Temme, K., Hill, R., Segall-Shapiro, T. H., Moser, F., & Voigt, C. A. (2012). Modular control of multiple pathways using engineered orthogonal T7 polymerases. Nucleic acids research, 40(17), 8773–8781.

Characterization

The Universal RBS was characterized by comparing it to BBa_B0032 . As a reporter, a GFP fluorescence readout was used. In order to measure fluorescence, we use a flow cytometer.
The GFP used as readout was our harmonized eGFP . All of the parts were cloned into a level 1 backbone pICH47761 .
The protocol for preparation of samples for the flow cytometry assay is as follows:

1. Samples were grown overnight
2. Overnight cultures were diluted to OD = 0.01 into 1 mL, and grow for 2 hours on 37 degrees 250 rpm shaking in 2 mL Eppendorf tubes.
3. Overnight cultures were diluted 1:100 into 5 mL, and grow for 4 hours on 37 degrees 250 rpm shaking in 50 mL eppendorf tubes. Induce with 1 mM IPTG where necessary
4. Samples were kept at 4 degrees for 1 hour

In the measurement, E. coli BL21 cells without a plasmid were used as a reference. The gating for flow cytometry was determined by eye by selecting the densest region of the blank. Furthermore, the fluorescence histogram was gated to discern between cells that were 'on' and 'off', as in expressing fluorescence or not. Only cells of similar forward and side scatter were compared. The median fluorescence intensity of the blank is subtracted from the fluorescence intensity of the samples to correct for autofluorescence. In figure 1 we plot the corrected fluorescence of the samples. Figure 2 shows the gating based on size and figure 3 shows the fluorescence histogram of each sample.

Figure 1: Fluorescence values of T7, T7sp1 with median fluorescence of E. coli BL21 cells without a plasmid substracted.

Figure 1 shows that the strength of our Universal RBS in E. coli BL21 is about 60% higher than BBa_B0032 .

Figure 2: Scatter plot of forward and side scatter of E. coli BL21 cells without a plasmid. The region selected is the gating we considered to obtain the values depicted in figure 1.

Figure 3: Raw fluorescence values. Black isE. coli BL21cells without a plasmid. Red is T7 and blue is T7_sp1