Coding

Part:BBa_K3885124

Designed by: Xue Li   Group: iGEM21_ZJUT-China   (2021-10-10)


σ28 (fliA)

σ28 is a special transcriptional factor concerning the fliA, which is an alternate sigma factor for the class 3 flagella operons.

Usage and Biology

Figure 1. Sequence logos for the -35 and -10 elements of the σ28-dependent promoter. (A) Sequence recognized by C. trachomatis σ28 RNA polymerase in the context of the C. trachomatis hctB promoter. (B) Sequence recognized by E. coli σ28 RNA polymerase in the same promoter context. (C) The sequence logo based on the nucleotide frequencies of known bacterial σ28 promoters.

σs have been divided into four groups based on their phylogenetic relationships and modular structure. σ28, a Group 3 σ, is the most widely distributed alternative σ factor , making it an attractive candidate for study of its promoter recognition. σ28 controls expression of flagella-related genes in all motile Gram-negative and Gram-positive bacteria, and plays a role in development in some non-motile bacteria.
Some researches found very similar results for σ28 RNA polymerase from C. trachomatis and E. coli, suggesting that promoter recognition by this alternative RNA polymerase is well conserved among bacteria. Bioinformatic analysis of E. coli s28 promoters suggests that their consensus sequence is TAAAgttt-N11-GCCGATAA.

Part uses:

Transcription is based on the core RNA polymerase and the primary sigma factor σ70 present in the cytoplasmic extract. All of the circuitries start with σ70 specific promoters. This σ70 E. coli promoter is the strongest so far reported. The six other sigma factors and the two bacteriophage RNA polymerases are expressed to engineer elementary gene circuits, such as transcriptional activation cascades and an AND gate. The repertoire of regulatory elements provided by σ70 specific promoters is much larger than bacteriophage systems. Yet, the transcription modularity with one sigma factor only is restrictive.

Figure 2.Crosstalk between transcriptional activation units measured in the linear regime of plasmid concentration. Values (deGFP [µM]) are from the end­point deGFP production for seven E. coli sigma factors (non­degradable versions).

The nonspecific gene expression generated through the promoter P70 by the alternative sigma factors could not be measured because the σ70 is present in the reaction. And we can see that σ28 and σ32 are the most competitive from the table. But, σ32 is the leakiest unit. The nonspecific expression through the promoter P32 is large even in the presence of the primary sigma factor only. Another important feature is the high specificity of σ28 and its promoter. The leak through the promoter P28 is almost systematically below the detection limit.So the σ28 unit is the most efficient and the most specific sigma factor unit tested in this work.

Figure 3. Schematic of cascade of gene circuits. σ28 is capable of motivating the promoter P28, and promote the expression of deGFP.

This year, ZJUT-China aimed to develop a biosensor to detect RNA biomarkers related to diseases. Cascade of genetic circuits was designed to be utilized in the biosensor with Cell-Free system. In the gene circuits, we constructed plasmid P70a-σ28, P28-tetO-deGFP, and P70-σ28-P28-tetO-deGFP.
The cascade of two plasmids was divided in the Cell-Free system, whereas two parts of the cascade were incorporated into one composite part in bacteria.

Characterization

P70-σ28+P28-tetO-deGFP

Figure 4. The standard curve of eGFP measured at 29℃, 488/535 nm Ex/Em, gain 60.

12μL Arbor bioscience myTXTL σ70 reagent carries plasmid P70a-σ28 (2nM), and P28-tetO-deGFP (10nM). After vortexing gently, pipette 5 μL from the reaction into two wells in the 96 V-bottom well plate. Seal the wells with caps, place the well plate into the plate reader, and begin measurement, incubate at 29℃, 10 minutes for 6h, 488/535 nm Ex/Em, at gain 60.

Result

Figure 5. Kinetics of deGFP expression in TXTL reactions carrying plasmid P70a-σ28 (2nM), and P28-tetO-deGFP (10nM).

The transcriptional factor σ28 combines with σ70 to become a cascade, which is more regulable than σ70 in the Cell-Free system. Meanwhile, σ28 is capable of activating the promoter P28 to express the deGFP gene, which is weaker than the P70a promoter.

Validation of σ28 in E. Coli BW25113

Figure 6. Schematic of the procedure of Electroporation-Transformation E. coli BW25113 cells .

To enhance the expression intensity of each component, we use σ28 as the promoter factor. σ28 is a special transcriptional factor concerning the fliA, which is an alternate sigma factor for the class 3 flagellar operons, and it does not exist in Cell-free system. To verify the functional availability of σ28, we build P70a-σ28-P28-deGFP plasmid. If the expression of σ28 is normal, it shows fluorescence. Conversely, there is no fluorescence. To verify the normal expression of σ28, we performed electroporation experiments with E. coli BW25113.

Result

Figure 7. Schematic of the flurescence intensity from E. coli BW25113. No plasmid is electroporated into E. Coli BW25113(a), plasmid P70a-σ28-P28-deGFP is electroporated into E. Coli BW25113(b), plasmid P28-tetO-deGFP is electroporated into E. Coli BW25113(c).

Figure 8. Schematic of fluorescence intensity from E. coli BW25113.

No colony in the control group fluorescenced (Figure 7 a) , while the single colony in the experimental group fluorescenced (Figure 7 b) , indicating that P70a-σ28-P28-deGFP was successfully transferred, all gene elements on the plasmid were functional, and σ28 was normally expressed. The measurement of fluorescence intensity from the plates is in Figure 8.

Model-σ28:

We built a deterministic gene expression model to analyze gene expression in the Cell-Free system. In this model, DNA transcription, mRNA degradation, and mRNA translation are described by Michaelis-Menten kinetics while protein maturation follows a first-order kinetics.


Equations

The system of equations describing the cascade P70a -σ28, P28-deGFP is as follows:

Details of the derivation process can be found at Model.

Parameter Determination

Catalytic rate constant for DNA transcription regulated by σ28

For optimal expression, P70a-σ28 and P28a-deGFP should be set at 0.5 nM and 15 nM, respectively. Due to the low concentration, we assume that P70a-σ28 provides sufficient σ28 without depleting the transcriptional and translational machinery. Thus, the maximum protein synthesis rate of P28-deGFP saturates at 15 nM. P70-deGFP reaches its maximal protein synthesis rate at 5 nM. So, we consider that the value of the catalytic rate constant for transcription regulated by σ28 is approximately one-third of that of σ70 :

Protein maturation rate constant of σ28

By fitting the model to the data from The all E. coli TXTL Toolbox 2.0, the protein maturation rate constant of σ28 is found to be 0.16 s-1 .

References

[1]Koo B. M. et al. Mutational analysis of Escherichia coli sigma28 and its target promoters reveals recognition of a composite -10 region, comprised of an 'extended -10' motif and a core -10 element.[J]. Molecular microbiology, 2009, 72(4) : 830-43.
[2]Yu H. et al. Mutational analysis of the promoter recognized by Chlamydia and Escherichia coli sigma(28) RNA polymerase.[J]. Journal of bacteriology, 2006, 188(15) : 5524-31.
[3]Shin J. and Noireaux V. An E. coli Cell-Free expression toolbox: application to synthetic gene circuits and artificial cells.[J]. ACS synthetic biology, 2012, 1(1) : 29-41.
[4]Marshall R. et al. Quantitative modeling of transcription and translation of an all-E. coli Cell-free system.[J]. Scientific reports, 2019, 9(1): 1-12.
[5] Garamella J, et al. The all E. coli TX-TL toolbox 2.0: a platform for Cell-Free synthetic biology.[J]. ACS synthetic biology, 2016, 5(4): 344-355.

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 451
    Illegal SapI.rc site found at 607
    Illegal SapI site found at 426


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