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In the measurement, <i>E. coli Top 10</i> cells without a plasmid were used as a blank. 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. </p>
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In the measurement, <i>E. coli Top 10</i> 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. </p>
  
 
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<html><body><figcaption><br><b>Figure 3: Raw fluorescence values. Black is <i>E. coli Top 10</i> cells without a plasmid. Pink is T7 and blue is Pbhr<sub>sp1 v2<sub> </b></figcaption></body></html>
 
<html><body><figcaption><br><b>Figure 3: Raw fluorescence values. Black is <i>E. coli Top 10</i> cells without a plasmid. Pink is T7 and blue is Pbhr<sub>sp1 v2<sub> </b></figcaption></body></html>
 
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===References===
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<a id="Segall2018" href="https://www.nature.com/articles/nbt.4111" target="_blank">
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Segall-Shapiro, T. H., Sontag, E. D., & Voigt, C. A. (2018). Engineered promoters enable constant gene expression at any copy number in bacteria. <i>Nature Biotechnology</i>, 36(4), 352–358.</a>
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Revision as of 15:54, 19 October 2019

Medium T7sp1 promoter

A T7 promoter variant with strength that should give 50% expression compared to T7 promoter. The T7 promoter variant contains a binding site for TALE 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]

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

Usage and Biology

The 0.5 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 be 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. 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 0.5 T7sp1 was characterized by comparing it to a T7 promoter. 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 Juniper GFP BBa_J97001 . The ribosome binding site was our Universal RBS . All of these 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 Top 10 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.

Modeling


Figure 1: Fluorescence corrected for autofluorescence of E. coli Top 10 cells without a plasmid.

Figure 1 shows that the strength of PBhr in E. coli is significantly higher than a T7 promoter induced using 1 mM IPTG for 4 hours. PBhrsp1 v2 contains the binding site for TALEsp1. The presence of the binding site seems to reduce the strength of the promoter.



Modeling


Figure 2: Scatter plot of forward and side scatter of E. coli Top 10 cells without a plasmid. The region selected is the gating we considered to obtain the values depicted in figure 1.

Modeling


Figure 3: Raw fluorescence values. Black is E. coli Top 10 cells without a plasmid. Pink is T7 and blue is Pbhrsp1 v2

References