Coding

Part:BBa_K3782005

Designed by: Jana Nf   Group: iGEM21_UNILausanne   (2021-10-17)
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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) RiAFP in the RiAFP-pColdI construct was visualized by a 1% Agarose Gel. Left to right: L - 1 kb DNA Ladder, 1 – pCold-I-RiAFP (588). (b) RiAFP in the pET-17b construct was visualized by a 1% Agarose Gel. Left to right: L - 1 kb DNA Ladder, 2 – pET-17b-RiAFP (640 bp).


RiAFP was cloned into the pET-17b plasmid using restriction and ligation via HindIII and XhoI and Gibson Assembly. The vector includes an ampicillin resistance gene and a lac operator, which allows for IPTG induced expression. The final construct was checked with a 1% Agarose gel (Fig.1) and via sequencing.

RiAFP was cloned into the pColdI plasmid using restriction and ligation via NdeI and XhoI and Gibson Assembly. The vector includes an ampicillin resistance gene and a lac operator, which allows for IPTG induced expression. The final construct was checked with a 1% Agarose gel (Fig.1) and via sequencing.












Expression and Purification

Figure 2:SDS-PAGE electrophoresis before and after RiAFP purification. RiAFP was expressed in E. coli BL21 (DE3) using both pCold-I and pET-17b vectors. Left to right: L - 10 to 250 kDa Protein Ladder, Bacterial Lysate: 1 - pET-17b-RiAFP, 2 - pCold-I-RiAFP. Purified Protein: 3 - pET-17b-RiAFP, 4 - pCold-I-RiAFP.


After we successfully transformed E. coli strain BL21 (DE3) with our pET-17b-RiAFPand pColdI-RiAFP constructs, we inoculated liquid cultures with LB and ampicillin. Before inducing expression in large volumes of liquid cultures, we first did small-scale test purifications to verify if the antifreeze proteins could be correctly purified. Next, we grew 1-liter liquid cultures at 37°C until an OD600 of 0.6 was reached. We then placed the culture in a cooling incubator at 15°C for 30 minutes. We added IPTG 1mM and induced expression at 15°C for 24 hours. Afterwards, we removed a small volume of culture to run a total cell SDS-PAGE (15%) of E. coli cultures stained with Coomassie blue and to verify that the antifreeze protein was correctly overexpressed. The remaining culture was centrifuged, and the cell pellet was resuspended in Cold Lysis Buffer. We sonicated the cells and centrifuged the cells once more to collect the supernatant, which we loaded into the His-tag affinity column. We eluted the antifreeze proteins which bound to the column and concentrated the solution to a smaller volume. We performed a gel filtration to further purify the protein and finally stored aliquots at -80°C.

We induced expression of RiAFP with IPTG and ran a total cell SDS-PAGE (15%) of the E. coli BL21 (DE3) cultures stained with Coomassie blue. We used two different vectors, pET-17b and pColdI, to compare how the promoters influenced expression efficiency. The overexpression was successful for RiAFP. We observed thicker bands that matched the weight of the protein. The pColdI vector worked better than the pET-17b vector, because the band appeared to be stronger for this first plasmid on the gel (Fig.2).


ISF Assay









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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