EC maynardl Fis

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Author Information

Lucy Maynard

Basic Information

  • ID: EG10317
  • Name: Fis
  • Organism: E. coli
  • UniProt ID: [1]
  • Description: Fis encodes for the FIS protein, a DNA-binding, ribosomal RNA transcription factor. FIS influences DNA replication and bacterial chromosome structure [1]. FIS acts as a trans-activator for more than 230 identified genes [2] This protein folds into a quadruple alpha-helix structure, with one beta-hairpin [3], and is most active during the exponential growth phase [4]. FIS is involved in maintaining and organizing the nuceloid envelope via direct DNA binding and subsequent modified gyrase activity[5]

I would be interested in collaborating with another student to incorporate FIS binding sites in the DNA sequence upstream of their assigned gene. Ideally this gene should be considered highly important to cell life, and 'worthy' of transcriptional enhancement. In E.coli, FIS binds to the DNA sequence 5' GATCTAGCTAATATTATTAGCTATAGCT 3'. I also find this protein interesting as it offers a structural benefit, and could initiate first steps of Shriram 1.0 DNA organization.

  • DNA Length: 297 base pairs.
  • DNA sequence:

ATGTTCGAACAACGCGTAAATTCTGACGTACTGACCGTTTCTACCGTTAACTCTCAGGATCAGGTAACCC AAAAACCCCTGCGTGACTCGGTTAAACAGGCACTGAAGAACTATTTTGCTCAACTGAATGGTCAGGATGT GAATGACCTCTATGAGCTGGTACTGGCTGAAGTAGAACAGCCCCTGTTGGACATGGTGATGCAATACACC CGTGGTAACCAGACCCGTGCTGCGCTGATGATGGGCATCAACCGTGGTACGCTGCGTAAAAAATTGAAAA AATACGGCATGAACTAA

  • Amino Acid length: 98 amino acids.
  • Amino Acid sequence:

MFEQRVNSDVLTVSTVNSQDQVTQKPLRDSVKQALKNYFAQLNGQDVNDLYELVLAEVEQPLLDMVMQYT RGNQTRAALMMGINRGTLRKKLKKYGMN

Function and Homologs

  • Product: FIS protein
  • Closest homologous proteins: The top (max three) homologous proteins to this protein, as identified by BLAST searches.

BLAST was taking >10min, so Uniprot BLAST tool was used:

    • DNA binding protein fis, 491/100%/3e-61/100%, [2]
    • DNA binding protein fis, 491/100%/3e-61/100%, [3]
    • DNA binding protein fis, 491/100%/3e-61/100%, [4]
  • Equivalent E. coli / JCVI functional protein: This is my extra-credit E.coli gene.

Expression

  • Expression Level: [low to medium].
  • Expression Level Hypothesis: Since FIS is an abundant protein in E.coli and is a strong activator of many genes, we should express it on a low to medium level, erring on the low side, since the outcome is completely unknown. No difference whatsoever may be observed.
  • Expression Level References and Description: Information was gathered from the following sources:

[6]


  • Expression Time: [early]
  • Expression Level Hypothesis: Transcriptional regulation is a complex process, so should be initiated once the most important energetic and metabolic demands have been met. However, in E.coli FIS has been shown to be most active during the exponential growth phase, in a high nutrient media [7]
  • Expression Time References and Description:

FIS EcoCyc: [5]

[8]

Gene Context

  • Other Components: 2 FIS monomers homodimerize to form the functional module.
  • Possible Dependencies: FIS has interesting autoregulatory mechanisms, such as self-induced DNA supercoiling via distal FIS binding site contact, and spatio-structural functions via nucleoid tethering. This means that FIS relies on DNA properties of bending and deformation to form supercoils, and the existence of a nucleoid in our synthetic system.
  • Process: Transcriptional activation
    • Inputs: FIS + FIS Binding site upstream of Gene of Interest (GOI)
    • Outputs: increased mRNA copies of GOI

Construct

We will handle this - not part of your assignment

  • Synthesis Score: The synthesis score: 1, 2,3
  • Predicted Translation Rate: Prediction of construct translation rate from the RBS calculator
  • Design Notes and Details:
  • GenBank File: A link to the GenBank file. file
  • Wold S, Crooke E, Skarstad K (1996)
  • Finkel, S. E., and R. C. Johnson. "The Fis protein: it's not just for DNA inversion anymore." Molecular microbiology 6.22 (2006): 3257-3265.
  • Steen JA, Steen JA, Harrison P, Seemann T, Wilkie I, et al. (2010) Fis Is Essential for Capsule Production in Pasteurella multocida and Regulates Expression of Other Important Virulence Factors. PLoS Pathog 6(2): e1000750. doi:10.1371/journal.ppat.1000750
  • Bradley, Meranda D., et al. "Effects of Fis on Escherichia coli gene expression during different growth stages." Microbiology 153.9 (2007): 2922-2940.
  • Schneider, Robert, et al. "An architectural role of the Escherichia coli chromatin protein FIS in organising DNA." Nucleic acids research 29.24 (2001): 5107-5114.
  • Ross, Wilma, et al. "E. coli Fis protein activates ribosomal RNA transcription in vitro and in vivo." The EMBO Journal 9.11 (1990): 3733.
  • Bradley, Meranda D., et al. "Effects of Fis on Escherichia coli gene expression during different growth stages." Microbiology 153.9 (2007): 2922-2940.
  • Condon, Ciaran, Craig Squires, and Catherine L. Squires. "Control of rRNA transcription in Escherichia coli." Microbiological reviews 59.4 (1995): 623-645.
  • Retrieved from "http://e80boot.org/index.php?title=EC_maynardl_Fis&oldid=15337"