Part:BBa_K2918040

_ T7 promoter based optimized iFFL

Genetic implementation of an incoherent feed-forward loop (iFFL) in which a low strength T7 promoter with a binding site for TALE 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
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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

An 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.

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.

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 neighboring transcriptonal unit can occur. This significantly influences the behavior of the iFFL genetic circuit (Segall-Shapiro et al., 2018). Hence, the transcriptional unit of TALE is oriented in a different orientation than the transcriptional unit of GFP.

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. To achieve expression robust to changes in transcriptional and tranlational rates, the ratio of transcriptional and translational rates of GFP and repressor need to be constant. This can be achieved by using orthogonal T7 promoter and its variants (T7 promoter, 0.5 T7 promoter, 0.1 T7 promoter, 0.5 T7sp1 promoter and T7sp1 promoter ).

Apart from being able to achieve stable gene expression across different bacterial species, it is necessary to attain different levels of gene expression. The T7 promoter based optimized iFFL can be used to obtain higher levels of gene of interest (GFP) expression as the expression of TALE is driven by a lower strength promoter (0.1 T7 promoter) compared to the promoter (0.5 T7sp1 promoter) driving GFP expression.

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

According to our model,the genetic implementation of the iFFL system yields the same gene of interest (GOI) expression levels when the transcription rate of both genes (TALE and GOI) are 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

For the system to be versatile, it is important to achieve different levels of output gene expression (GFP). Hence, in the optimized iFFL system the TALE repressor is expressed by a higher strength promoter compared to the promoter controlling GFP expression.

To validate the insulation of the gene expression (GFP) to changes in transcriptional rates, GFP flourescence was measured at different IPTG concentrations by flow cytometry at log-phase of growth. Change in IPTG concentrations, changes in in-vivo concentrations of T7 RNAP and this contributes to variations in transcriptional rates. In unrepressed systems, the expression of the GOI is a function of IPTG concentrations. However, in iFFL systems, since the transcriptional rates of TALE and GFP are under the control of T7 promoters, similar GOI expression is expected. As a control we expressed GFP under the control of T7sp1 promoter was used. As a reference for background fluorescence, E.coli BL21 DE(3) cells without a plasmid was used. The protocol for sample preparation can be found here,

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Scatter plots (figure 4 and 5) for the unprepressed control and T7 based optimized iFFL was gated by eye to select for the most dense regions.

Figure 4: Scatter plot for unrepressed control (forward scatter vs side scatter)

Figure 5: Scatter plot for T7 based optimized iFFL system (forward scatter vs side scatter)

Figures 6 and 7 show fluorescence measurements for the gated populations of the unrepressed control and optimized iFFL system at different IPTG concentrations.

Figure 6: Flourescence measurements from gated population of unrepressed control at different IPTG concentrations. Black: E.coliBL21 DE(3), Green: 0mM, Red:0.1mM, Blue:0.5mM and Purple: 1mM

Figure 7: Flourescence measurements from gated population of unrepressed control at different IPTG concentrations. Black: E.coliBL21 DE(3), Green: 0mM, Red:0.1mM, Blue:0.5mM and Purple: 1mM

In figure 6, it is seen that GFP fluroscence of the unrepressed control changes with IPTG concentrations. However,(in figure 7) for the optimized iFFL system, the GFP fluroscence curves for all the IPTG concentrations overlap and therefore is independent of IPTG concentrations.

In figure 8, the median fluroscence for the unrepressed control and optimized iFFL system are plotted at different IPTG concentrations.

Figure 8: Steady state GFP flourescnece measurements using flow cytometry at different IPTG concentrations. Measurements were made at log-phase of growth and the median flourescence of the gated populations is plotted.

From figure 8, it is seen that despite changes in IPTG the GFP fluroscence remains the same while the GFP fluroscence of the control increases with IPTG concentrations. This suggests that the gene of interest expression is independent of IPTG concentrations thereby independent of transcriptional rate.

Conclusion

The characterization of the T7 based optimized iFFL system suggests that the circuit is robust to changes in transcriptional rates.

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.