Part:BBa_K3885124

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 flflagella-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. </p>

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 most leaky 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 were 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Unknown
• 21
  INCOMPATIBLE WITH RFC[21]

  Unknown
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

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