Device

Part:BBa_K2940000:Design

Designed by: Mirren White   Group: iGEM19_Edinburgh_OG   (2019-10-02)


Zinc-sensitive Biosensor producing CotA Laccase


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1988
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 327
    Illegal BamHI site found at 1745
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 665
    Illegal NgoMIV site found at 1559
    Illegal AgeI site found at 199
    Illegal AgeI site found at 362
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1017


Design Notes

The original promoter-operator sequence from the paper contained an overlap of the beginning of the smtB coding sequence, which prevented synthesis due to a hairpin loop. These bases were removed.

The sensor is constructed of several discrete parts, each of which has its own particular function within the circuit. Each part, and its function, is described below .

SmtB is the circuit controller. It is a small (122 amino acids) dimeric protein that acts as a transcriptional repressor through direct binding with DNA. The sequence was taken from the genomic data for S. elongatus strain PCC7942 (GenBank ID: X64585) and includes the portion upstream of the coding sequence.

In S. elongatus, between the smtB and smtA genes lies a short bidirectional promoter sequence. Here, a 111bp portion of this sequence that contains the recognition site for the SmtB repressor protein is used to control expression from both genes in the circuit. In previous research on this segment, a 141bp [10] length was used. Here, the length of this portion has been shortened by removing several bases that overlap the beginning of the smtB gene from the previous sequence.

BBa_B0030 is a BioBrick part which encodes a strong ribosomal binding site [12]. This sequence was included to maximise the expression of the protein. Sequence: ATTAAAGAGGAGAAA

PelB, originating from Erwinia carotovora, is a short (22aa) leader sequence which is attached to the N-terminal of the coding sequence of both laccase and the positive control. This was intended to ensure the produced protein is exported to the cell membrane, where the leader sequence is then cleaved by PelB peptidase, and the protein is secreted. The pelB sequence was taken from an existing BioBrick, BBa_J32015 [13].

PelB was included due to the results obtained by a previous iGEM team in 2018, which showed an increase in laccase activity in both cell lysate and supernatant when PelB was fused to the CotA coding sequence [14]. This construct improves on their BioBrick by the inclusion of restriction sites for ApaI and NdeI flanking the enzyme coding sequence. This allows the enzyme to be removed and replaced with another coding sequence, in this case to construct a positive control.

CotA laccase is a well-characterised classical laccase enzyme from Bacillus subtilis. It was chosen as previous iGEM teams have worked with this enzyme and there is significant literature to support its ability to degrade a variety of azo dyes. This sequence was taken from the B. subtilis genome sequence (GenBank ID: GU972589). A single base pair substitution was made to remove a PstI restriction site in the coding sequence.

EiraCFP is an open-source cyan fluorescent protein which is used here as a positive control. The protein itself is colourless, which should avoid any issues with the colour interfering with spectrophotometric measurements. This sequence is taken from a BioBrick part, BBa_J97000 [15].

Source

S elongatus genomic sequence, B subtilis genomic sequence

References

[1]Bhardwaj V, Kumar P, Singhal G. Toxicity of Heavy Metals Pollutants in Textile Mills Effluents. Int J Sci Eng Res. 2014;5: 664–666. Available: https://www.ijser.org/researchpaper/Toxicity-of-Heavy-Metals-Pollutants-in-Textile-Mills-Effluents.pdf

[2] Tay PKR, Nguyen PQ, Joshi NS. A Synthetic Circuit for Mercury Bioremediation Using Self-Assembling Functional Amyloids. ACS Synth Biol. American Chemical Society; 2017;6: 1841–1850. doi:10.1021/acssynbio.7b00137

[3]Kenzom T, Srivastava P, Mishra S. Structural insights into 2,2’-azino-Bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)-mediated degradation of reactive blue 21 by engineered Cyathus bulleri Laccase and characterization of degradation products. Appl Environ Microbiol. 2014/09/26. American Society for Microbiology; 2014;80: 7484–7495. doi:10.1128/AEM.02665-14

[4] Morita EH, Wakamatsu M, Uegaki K, Yumoto N, Kyogoku Y, Hayashi H. Zinc Ions Inhibit the Protein–DNA Complex Formation between Cyanobacterial Transcription Factor SmtB and its Recognition DNA Sequences. Plant Cell Physiol. 2002;43: 1254–1258. doi:10.1093/pcp/pcf140

[5] Hussein F. Chemical Properties of Treated Textile Dyeing Wastewater. Asian Journal of Chemistry. 2013. doi:10.14233/ajchem.2013.15909A

[6] Yaseen DA, Scholz M. Textile dye wastewater characteristics and constituents of synthetic effluents: a critical review. Int J Environ Sci Technol. 2019;16: 1193–1226. doi:10.1007/s13762-018-2130-z

[7] Busenlehner LS, Pennella MA, Giedroc DP. The SmtB/ArsR family of metalloregulatory transcriptional repressors: structural insights into prokaryotic metal resistance. FEMS Microbiol Rev. 2003;27: 131–143. doi:10.1016/S0168-6445(03)00054-8

[8] Cavet JS, Meng W, Pennella MA, Appelhoff RJ, Giedroc DP, Robinson NJ. A Nickel-Cobalt-sensing ArsR-SmtB Family Repressor: CONTRIBUTIONS OF CYTOSOL AND EFFECTOR BINDING SITES TO METAL SELECTIVITY . J Biol Chem . 2002;277: 38441–38448. doi:10.1074/jbc.M207677200

[9]National Center for Biotechnology Information. Escherichia coli str. K-12 substr. MG1655 strain K-12 chromosome [Internet]. 2018 [cited 29 Jul 2019]. Available: https://www.ncbi.nlm.nih.gov/nucleotide/CP027060.1?report=genbank&log$=nuclalign&blast_rank=88&RID=KWCFA324014&from=3284213&to=3284307

[10]Zn2+-Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor Adam A. Pérez, John P. Gajewski, Bryan H. Ferlez, Marcus Ludwig, Carol S. Baker, John H. Golbeck, Donald A. Bryant Applied and Environmental Microbiology Jan 2017, 83 (3) e02491-16; DOI: 10.1128/AEM.02491-16

[11]Erbe JL, Taylor KB, Hall LM. Metalloregulation of the cyanobacterial smt locus: indentification of SmtB binding sites and direct interaction with metals . Nucleic Acids Res. 1995;23: 2472–2478. doi:10.1093/nar/23.13.2472

[12]Mahajan VS, Marinescu VD, Chow B, Wissner-Gross AD, Carr P. Part:BBa_B0030 - RBS.1 (strong) -- modified from R. Weiss [Internet]. 2003. Available: https://parts.igem.org/Part:BBa_B0030

[13]Heinz A, Duke University iGEM Team. Part:BBa_J32015 [Internet]. 2006. Available: https://parts.igem.org/Part:BBa_J32015

[14]2018 S iGEM T. Part:BBa_K2684001 [Internet]. 2018 [cited 13 Jul 2019]. Available: https://parts.igem.org/Part:BBa_K2684001

[15]Endy D, BioFAB. Part:BBa_J97000 - IP-Free EiraCFP (Cyan Fluorescent Protein) [Internet]. 2014. Available: https://parts.igem.org/Part:BBa_J97000