Part:BBa_K2918048

Medium T7 promoter based iFFL

Genetic implementation of an incoherent feed forward loop (iFFL) in which a stabilized medium 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

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 (as shown in Figure 1).

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 (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 protein represses GFP

When transcriptional units are placed in series, due to low efficiency of terminators, leaky expression of the gene in the neighboring transcriptional 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 (as shown in Figure 2) 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 transcriptional 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, Weak T7 promoter, Medium T7 promoter, Medium 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 BsaI based golden gate assembly. The multi-transcriptional unit construct was assembled by a BpiI 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.

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 loop 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

Different transcriptional rates can be achieved by using T7 promoter and its variants (Komura et al., 2018). To demonstrate stable gene expression (GFP), iFFL loops were constructed using two T7 promoter variants and the corresponding promoters with TALEsp1 binding sites. Two circuits were constructed, one where TALE and GFP are expressed with wild-type T7 and T7sp1 promoters (T7 promoter based iFFL) respectively and the other where the two genes are expressed by medium T7 and medium T7sp1 promoters respectively.

During initial verification of the iFFL loop, unrepressed iFFL (i.e GFP was expressed with a promoter not containing the TALEsp1 binding site) was used as control. Unexpectedly, low fluorescence was observed for this control. From flow cytometry results (Figure 4) it was seen that the cells containing the unrepressed iFFL system were smaller in size and had different cell morphology (region R1) than E. coli cells without any plasmid (region R0) and therefore can not be compared to each other. The difference in cell morphology suggests that over expression of TALE and GFP could be burdensome to the cells. Hence, for further data analysis, GFP expressed from T7_sp1 was used as a control. E. coli BL21 (DE3) were used to correct for background fluorescence.

Figure 4: Scatter plot of forward (FSC) and side (SSC) scatter of E. coli and the unrepressed iFFL system. R0 is the densest region of E. coli and R1 the densest region of the unrepressed TALE system

The scatter plot in Figure 5 was used to gate the most dense cell regions of the blank and the same gating was considered to obtain the fluorescence values depicted in Figure 6. Cells of similar forward and side scatter were compared.

Figure 5: Scatter plot of forward and side scatter of E. coli BL21(DE3) cells without a plasmid. The region selected is the gating we considered to obtain the values depicted in Figure 6.

Gating was performed on the data in the fluorescence histogram (Figure 6) to discern between fluorescent and non-fluorescent cells.

Figure 6: Raw fluorescence values of E. coli BL21 (DE3) cells without a plasmid (black), unrepressed iFFL (grey), medium T7 based iFFL system (blue), T7 based iFFL system (purple) and control (Green)

The median fluorescence for each sample from Figure 6 was plotted for comparison in Figure 7.

Figure 7: GFP fluorescence measured during logarithmic growth phase. Median fluorescence values derived from Figure 6 corrected for background fluorescence.

Figure 7 shows GFP expression levels (measured during logarithmic growth phase) of T7 based iFFL and medium T7 based iFFL. Despite change in transcriptional rates, similar GFP expression is observed.

Conclusion

Results above indicate successful implementation of the iFFL system to insulate from transcriptional variations. Transcriptional variations were achieved by using T7 promoters of different strengths. Despite changing promoters, the same GFP expression is observed this is because the ratio of transcriptional rates (promoter strengths) of TALE and GFP are similar in both the T7 based iFFL and medium T7 based iFFL.