Part:BBa_K2918046

T7 promoter based iFFL

Genetic implementation of an incoherent feed forward loop (iFFL) in which a stabilized T7 promoter is controlling GFP expression.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 283
  Illegal PstI site found at 2538
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 283
  Illegal PstI site found at 2538
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 250
  Illegal XhoI site found at 3335
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 283
  Illegal PstI site found at 2538
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 283
  Illegal PstI site found at 2538
  Illegal AgeI site found at 1277
  
• 1000
  COMPATIBLE WITH RFC[1000]

The two transcriptional units in this composite part are oriented outwards.

Usage and Biology

A Incoherent feed-forward loop (iFFL) is a unique control systems motif where the output signal is robust to changes in the input signal. This is achieved by the introduction of a repressor.

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  Figure 1: Overview of incoherent feed-forward loop

iFFL can be applied to genetic circuits to achieve expression independent from copy number, transcriptional and translational rates. To implement the iFFL in a genetic circuit, TALE proteins can be used. These proteins consist of repeats where 12th and 13th amino acids can vary, these are called the repeat variable diresidue (RVD). RVDs have been shown to bind to DNA in a simple one-to-one binding code (Doyle, Stoddard et al., 2013). The direct correspondence between amino acids allows scientists to engineer these repeat regions to target any sequence they want. In our system, we used the TALE protein as a repressor by engineering promoters to contain the binding site of this specific TALE protein (0.1 T7sp1 promoter, 0.5 T7sp1 promoter and P_BHRsp1 promoter).
In our genetic circuit, a unrepressed promoter controls the expression of TALE protein while the promoters with the TALE binding sites drive expression of GFP.

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  Figure 2: Overview of how the TALE proteins represses GFP

When transcriptional units are placed in series, due to low effieciency of terminators, leaky expression of the gene in the neighbouring transcriptonal unit can occur. This significantly influences the behavior of the iFFL genetic circuit (Segall-Shapiro et al., 2018). Hence, the transcriptional units in the circuit are oriented outward to achieve insulation from influence of the neighboring transcriptional unit.

An interesting application of the iFFL is to achieve controllable gene expression across different bacterial species. Gene expression in different bacterial contexts is influenced by changes in copy number, transcriptional and translational rates. Gene expression independent of transcrioptional rates has been demonstrated below. Stable gene expression can be attained if the ratios of transcriptional rates of GFP and repressor are constant. Transcriptional rates can be tuned using orthogonal T7 promoter and its variants (T7 promoter, 0.5 T7 promoter,0.1 T7 promoter, 0.5 T7sp1 promoter and T7sp1 promoter ).

Strain Construction

The construct was assembled by golden gate assembly based modular cloning system. First, the individual transcriptional units were cloned into level 1 destination vectors pICH47761 and pICH47822 by BpiI based golden gate assembly. The multi-transcriptional unit construct was assembled by a BsaI based golden gate. The assembly was a one-pot restriction-ligation reaction where the individual level 1 constructs were added along with the destination vector pICH48055. The correct clone was distinguished by blue-white screening and the construct 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). 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

Characterization

We have characterized the behavior of this system under the induction of different concentrations of IPTG. According to our model this system always yields the same gene of interest (GOI) expression levels when the transcription rate of both genes (TALE and GOI) is changed in the same ratio as can be seen in figure 3.

Figure 3: Steady-state GFP production while transcription rates of both TALE and GOI are changed. The lines indicate constant ratio of transcription rates

In order to measure steady-state GFP levels we measured fluorescence in log-phase using flow cytometry.

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.
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 for background fluorescence. As a control, harmonized eGFP driven by the same promoter and RBS was used. The gating for flow cytometry was determined by eye by selecting the densest region of E. coli TOP10. 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 6 we plot the corrected fluorescence of the samples. Figures 7 and 8 show the gating and the fluorescence histogram of each sample for the negative control. Figures 9 and 10 show the same but for our optimized stabilized iFFL systems. </p>

Figure 6: Steady-state GFP fluorescence measurement of promoter variation using FACS. The graph depicts T7 and 0.5 T7 iFFL systems, expected to give the same fluorescence according to the model. As a control, GFP under control of an unrepressed T7 promoter was used.

Figure 6 shows that our T7 based TALE system results in the same expression when both promoters are changed to their medium version. We expected the unrepressed T7 system to have higher expression similarly to the GFP under control of T7 without TALE expressed. However, as shown in figure 6 this is not the case. Although, when comparing the scatter plots of both E. coli TOP10 and the unrepressed T7 system we can see there is a clear difference in forward scatter (FSC) and side scatter (FSC) (figure 9), we believe the overexpression of both proteins significantly affects cell morphology and thus can't be properly compared to the other samples.

Figure 7: Scatter plot of forward and side scatter of E. coli TOP10 cells without a plasmid. The region selected is the gating we considered to obtain the values depicted in figure 6.

Figure 8: Raw fluorescence values of our stabilized and unrepressed systems. Black isE. coli BL21cells without a plasmid. Grey is TALE system without binding site for TALE in promoter controlling GFP. Blue is the medium T7 system and purple is the T7 system. Green is GFP controlled by T7 without any TALE expressed.

Figure 9: Scatter plot of forward (FSC) and side (SSC) scatter of E. coli and the uncontrolled TALE system. R0 is the densest region of E. coli and R1 the densest region of the uncontrolled TALE system

References

• Lou, C., Stanton, B., Chen, Y.-J., Munsky, B., & Voigt, C. A. (2012). Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nature Biotechnology, 30(11), 1137–1142.