Coding

Part:BBa_K3782005

Designed by: Jana Nf   Group: iGEM21_UNILausanne   (2021-10-17)
Revision as of 12:49, 17 October 2021 by Jana1907 (Talk | contribs)

Rhagium inquisitor ice-binding protein gene

RiAFP

RiAFP is a protein coding region that codes for an antifreeze protein (AFP). AFPs can bind to ice crystals and thereby prevent further ice growth. They are produced by organisms to survive in extremely cold environments. Activities of AFPs can be characterized by their thermal hysteresis (TH) or by their ice recrystallization inhibition (IRI). TH activity corresponds to the lowering of the freezing point without changing the melting point of a solution. IRI activity inhibits the growth of large ice crystals at the expense of smaller ones. The combination of these activities, which vary depending on the protein structure, prevents the freezing of body fluids and cell damage in organisms that live in environments with extremely cold temperatures.



Profile

Name RiAFP
Base pairs 405
Number of amino acids 134
Molecular weight 12.54 kDa
Origin Rhagium inquisitor, synthetic

Usage and Biology

The RiAFP protein coding region was used in the following composite parts (add links). It was expressed in E. coli strain BL21 (DE3) and purified. Various tests and assays were performed to characterize and verify the functionality of this ice-binding protein. RiAFP has moderate TH and IRI activity, and it can bind to ice crystals and inhibit their growth. The aim of our project was to use it on its own or in a mixture of other antifreeze proteins to develop a solution which could be applied on sensitive plant tissues and thereby protect crops from frost damage.

RiAFP is naturally produced by the Siberian cerambycid beetle Rhagium inquisitor. Its TH activity is around 6 K[1].

Antifreeze proteins bind to ice crystals and inhibit their growth. Ice crystals don’t have the same molecular arrangement on each of its faces and certain AFPs tend to adsorb to the basal plane of the crystals whilst others tend to adsorb to the prism plane. Hyperactive AFPs can bind to every plane[2]. The mechanism by which AFPs suppress the freezing point of a solution still isn’t completely understood. Nevertheless, AFPs are believed to attach irreversibly to the plane of a growing ice crystal. As the protein adsorbs to the growing ice plane, growth at the site is suppressed, producing bulges in between the adsorbed proteins[3]. Due to the Gibbs-Thomson effect, the freezing point will be depressed[4].

Characterization

To reduce frost damage during late spring freeze, we wanted to develop a solution containing antifreeze proteins, which bind to ice crystals and inhibit their growth. RiAFP was therefore chosen as one of three AFPs, cloned and expressed in E. coli BL21 (DE3), and finally purified using a His-tag affinity column and gel filtration.
Two different vectors were used for cloning: pET-17b and pColdI, containing a T7 promoter and a cold-shock protein A (cspA) promoter respectively.

Cloning

Figure 1: (a) FfIBP in the FfIBP-pColdI construct was visualized by a 1% Agarose Gel. Left to right: L - 1 kb DNA Ladder (N3232), 1 – FfIBP colony PCR with pColdI-FPrimer and pColdI-RPrimer. (b) FfIBP in the pET-17b construct was visualized by a 1% Agarose Gel. Left to right: L - 1 kb DNA Ladder (N3232), 2 – FfIBP colony PCR with pET17b-FPrimer and pET17b-RPrimer.







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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 130
    Illegal AgeI site found at 163
  • 1000
    COMPATIBLE WITH RFC[1000]


  1. ↑ Hakim A, Thakral D, Zhu DF, Nguyen JB. Expression, purification, crystallization and preliminary crystallographic studies of Rhagium inquisitor antifreeze protein. Acta Crystallogr Sect F Struct Biol Cryst Commun [Internet]. 2012 May [cited 2021 Oct 8];68(Pt 5):547. Available from: /pmc/articles/PMC3374510/
  2. ↑ Olijve LLC, Meister K, DeVries AL, Duman JG, Guo S, Bakker HJ, et al. Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins. Proc Natl Acad Sci [Internet]. 2016 Apr 5 [cited 2021 Oct 10];113(14):3740–5. Available from: https://www.pnas.org/content/113/14/3740
  3. ↑ LM S, AV T. Kinetic pinning and biological antifreezes. Phys Rev Lett [Internet]. 2004 Sep 17 [cited 2021 Oct 10];93(12). Available from: https://pubmed.ncbi.nlm.nih.gov/15447309/
  4. ↑ Pereyra RG, Szleifer I, Carignano MA. Temperature dependence of ice critical nucleus size. J Chem Phys [Internet]. 2011 Jul 21 [cited 2021 Oct 10];135(3):034508. Available from: https://aip.scitation.org/doi/abs/10.1063/1.3613672
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