Team:UPCH Peru/Design


In order to solve this problem, we turned to antifreeze proteins (AFPs). These proteins confer freezing resistance and freezing tolerance to organisms that live in cold environments [1]. As a result, our first objective was to select proper AFPs and obtain them via a reliable expression system. Additionally, working with AFPs demanded we understood the basis of how freezing occurs. Thus, our second objective was to comprehend, through testing, freezing and antifreezing activities. Finally, one of our goals is for these AFPs to be readily available in areas affected by frost on crops. Accordingly, our expression system needs to be able to work in cold temperatures; and for that we needed to test the viability of a candidate cold-tolerant organism.

  1. Selection and expression of antifreeze proteins (AFPs)

  2. Understand freezing and antifreeze activity

  3. Characterization of Pseudoalteromonas nigrifaciens

Selection and expression of antifreeze proteins(AFP)

In order to select the proper AFPs for our work, we need to understand the different properties they possess. AFPs have two mechanisms to prevent freezing. The first mechanism confers resistance to freezing by lowering the freezing temperature and slightly increasing the melting temperature of water, the difference between the new melting and freezing points is known as thermal hysteresis (TH), this mechanism is strong in AFPs from freeze-resistant organisms [1]. The second mechanism prevents growth of already formed large ice crystals and is called ice recrystallization inhibition (IRI), this mechanism is often present in AFPs from freeze-tolerant organisms [2].

TH and IRI activity are present in all AFPs but to different degrees. Our first option was to favor AFPs with a strong TH activity like the AFP from Tenebrio molitor, TmAFP. At a concentration of 1 mg/ml, it can make TH reach 4 °C [3,4]. Even though TmAFP has a strong TH activity, its purification could be problematic [5]; furthermore, since it comes from an insect and its IRI activity is low [3,4], there is a possibility that its antifreeze mechanisms may not be entirely fit for plants. Therefore, we turned to AFPs from the perennial ryegrass Lolium perenne: LpAFP and LpIRI3, which have a high IRI activity but a low TH activity (only 0.08 TH from 0.01 mg/ml) [6]. These proteins have conferred freezing tolerance in transgenic Arabidopsis thaliana and showed great activity when expressed in E. coli [6].

Once we had these three options in mind, we started to wonder how we were going to go about obtaining these AFPs. Consequently, we began with a design fit for expressing them in E. coli. The first design we came up with involved three main components: a commonly used inducible promoter (induced by IPTG), either of the three AFPs and the T7 terminator. This design was meant for expression of AFPs in E.coli

Nevertheless, our goal is to be able to express these AFPs in a cold environment. As a result, we needed an organism able to thrive in these conditions, such as Pseudoalteromonas nigrifaciens. This bacteria has a periplasm to which proteins can be transported, via a signal peptide, for purification purposes [7]. Moreover, different promoters work better in it given that they are from the Pseudoalteromonas genus [8,9]. Consequently, we needed a redesign to accomplish this.

The new design consisted of one of three possible new promoters (two inducible and one constitutive), a signal peptide, either of the three AFPs and the TaspC terminator. The inducible promoters only work in a specific culture media each. Then, through a vector fit for P. nigrafaciens, transform the organism and express the AFPs.

Why Pseudoalteromonas nigrifaciens?

We knew that E. coli, despite being a good expression vehicle, could not produce our protein in the most efficient way at such low temperatures [10]. Therefore, we decided to look for another chassis that meets the following characteristics:

  • Resistance to low temperatures compatible with frost.
  • Be classified as a BSL-1 organism
  • Used before as an expression system.
  • Could grow in conditions of high salinity, in which there is a low probability of contamination and thus facilitates the AFP production process.
  • Do not have many requirements for its maintenance.

Investigating the most suitable organism for our experimental design, we found several articles in which it was indicated that a genus of marine bacteria, called Pseudoalteromonas, had the characteristics that interested us [11-13]. One of the bacteria, Pseudoalteromonas haloplanktis, has been widely characterized in the literature [11-17]. However, for economic reasons, a phylogenetically close specie was chosen: Pseudoalteromonas nigrifaciens [17,18].

P. nigrifaciens is a gram-negative marine bacterium. It is rod-shaped and has a polar flagellum. It can grow in a wide temperature range and is characterized by producing melanin pigments at temperatures from 4°C [17-19]. This last feature facilitates its identification process at very low temperatures. Likewise, since it is marine, it can survive in environments with high salinity [19], which reduces the possibility of contamination. In addition, according to the ATCC®, it is classified as a biosafety level 1 organism, which makes it a safe organism to work with in the laboratory. There are few studies in which this bacterium has been used as a expression system, which is why in the first place, the following objectives were sought:

First, it was decided to work with E. coli as an expression system, since having more knowledge of its use in protocols, it would serve in a pilot experiment, as well as for the development of protein activity assays at the laboratory. Subsequently, the final chassis would be worked on, which will be placed in the bioreactor.

Understanding freezing and antifreeze activity

The melting and freezing point of water is 0 ° C (273.15 K) [20,21]. However, for this to happen, the nucleation process must be favored, which consists of the union of small ice nuclei [22]. This nucleation can be homogeneous or heterogeneous. The homogeneous nucleation of pure water consists of exceeding the high activation energy, and the temperature must be close to -40 °C for it to occur spontaneously [23,24]. On the other hand, heterogeneous nucleation is a process catalyzed by particles present in the system. These particles are called nucleating agents. Examples of nucleating agents are dust, some soluble salts, INA bacteria, among others [23-25].

For counteracting this phase change, there are different types of antifreeze agents, which can be of chemical or biological origin. Some chemical antifreeze agents are sodium chloride, magnesium chloride, ethylene glycol, among others [26]. Their function is to prevent freezing by modifying thermodynamic factors such as entropy and Gibbs free energy [27]. Instead, IBPs adhere to the surface of an ice crystal and follow an adsorption-inhibition mechanism [28].

Both chemical antifreeze agents and antifreeze proteins (AFPs), a type of IBPs, can lower water's freezing temperature. However, the chemical antifreeze agents do it in a colligative manner. It means that the number of solute particles added increases the entropy of the system, requiring more energy to be removed for the system to pass to an ordered state [27]. In contrast, AFPs do not [29].

With this theoretical basis, it was decided to carry out freezing experiments of distilled water at subzero temperatures in the presence and in the absence of nucleating agents. Likewise, it was decided to carry out freezing experiments of aqueous solutions of chemical antifreeze agents in the presence of a nucleating agent. In this way, the phenomena of freezing and antifreeze can be shown qualitatively, the latter being the one expected from AFPs.


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