Characterization of the bacteria
Since Pseudoalteromonas nigrifaciens is a bacteria that has not been widely used in the field of synthetic biology, we wanted to characterise deeper its growth and which factors would affect it. First, we did a bibliographic revision in which we could collect different aspects of our chassis. For it, we found the following information:
This is a gram negative rod-shaped marine bacteria. It has a wide range of growth temperature, being inactivated at temperatures higher than 30°C - 37°C [7-9]. It is strictly aerobic and can survive to salt concentrations between 1% to 10% [8]. It can produce melanin pigmentation when growing at 4°C and could be inactivated if the medium contains ascorbic acid, EDTA or L-cysteine [9]. Does not need any additional growth factors. Also, it can metabolise citrate and sorbitol, has DNAse activity and motility due to its polar flagellum, among others [7-9]. Depending on the strain, its antibiotic susceptibility may vary. According to ATCC®, this bacteria is categorized as BSL-1. It also could grow at depth [6].
Growth medium
According to a few scientific articles characterising the Pseudoalteromonas genre[6,8,9], the most suitable culture medium for P. nigrifaciens was the Marine broth Difco® 2216 (BD 279110). However, for the purpose of our project, we needed to create a simple medium in which our bacteria could grow optimally and be able to produce our protein in an efficient way. After trying to recreate this marine medium without success, due to the high amount of different salts needed and not efficient bacterial growth, we found another medium called Pseudomonas bathycetes medium that was suitable as well for P. nigrifaciens [10]. It consists of high concentrations of sodium chloride, proteose peptone, magnesium sulfate heptahydrate, magnesium chloride, yeast extract, and potassium chloride [10]. For its preparation, the composition of the LB medium was adapted and the additional salts concentrations were added. As it was prepared like broth and as a solid medium, it did not present precipitation even after these were autoclaved. After that, we proceeded to culture our bacteria by streak method, adding 20uL of the previous medium in which we activated our bacteria. Then, we let it grow again overnight at 25°C degrees. Compared to the first recreated medium, the new one had a major bacterial growth and the brownish colour of the colonies were clearer.
We also could corroborate the selectivity of this medium through the culturing of three different bacteria: E. coli, Salmonella spp and Pseudomonas, besides P. nigrifaciens. As a result, the only bacteria that could survive at this high salt concentration was the latter. Thus, we could assure that this medium would avoid any possibility of accidental contamination.
Electroporation protocol for P.nigrifaciens
After reviewing the literature, we realized that the electroporation method, using the genus Pseudoalteromonas, has a low transformation efficiency (102 - 105 CFU / ug DNA) [11,14]. One of the main reasons is that this genus grows in high salt concentrations [15,16], which acts as a barrier, preventing the binding of exogenous DNA to the cell surface [16]. Furthermore, the abundant presence of restriction-modification systems, in the Pseudoalteromonas genome, decreases the efficiency of DNA internalization, since these systems degrade foreign DNAs [15-16]. On the other hand, containing multiple resistance genes and drug efflux pumps allows cells to survive antibiotic pressure; therefore, hindering the counter-selection of transformants [15-16].
Likewise, contradictory results have been reported when handling strains of Pseudoalteromonas [16]. While in the study by Zhao et al. [13] it was possible to efficiently transform by electroporation the plasmid pWD2 into a strain of Pseudoalteromonas, Wang et al. [15] could not electroporate the same plasmid in other strains of the same genus. In contrast, in the latter study, the plasmid pWD2-oriT could be transferred by conjugation with Escherichia coli as donor cells [15]).
Given that the DNA transferred by conjugation is single-stranded, unlike electroporation, this could reduce the possible degradation by restriction-modification systems that preferably degrade double-stranded DNAs [15,17]. As a result, conjugation techniques have been widely used for genetic manipulations in Gram negative bacteria, such as Pseudoalteromonas [15,18].
However, the conjugation method is still time-consuming and certain problems can arise when selecting transconjugants after mating [16]. Therefore, we have chosen to transform P.nigrifaciens using electroporation. This species, specifically, does not have its own electroporation protocol, nor has it been transformed by any other method. However, other species and strains of Pseudoalteromonas have been used in electroporation and conjugation [11-15].
Within the investigated electroporation protocols, we have chosen the one proposed by Delavat et al. [14], since it’s a rapid and efficient protocol to introduce exogenous DNA in Pseudoalteromonas sp. A small amount of plasmid DNA (200 ng) was enough to obtain 50-60 transformed colonies, which means that a transformation efficiency of at least 1.25 x 102 CFU / ug DNA was obtained [14]. Although this is not the highest efficiency, it remained constant when electroporating two different strains of Pseudoalteromonas sp., while other protocols only worked with one strain (6-8). This suggests that, by using Delavat’s protocol [14], we could obtain approximately the same efficiency by working with a different strain of the same genus (P. nigrifaciens strain 217). Furthermore, the protocol includes a wash buffer with 15% glycerol to avoid cell lysis, and allowing to quickly freeze competent cells in liquid nitrogen and store them at -80ºC until later use [14]. In the same article, it is mentioned that the cells kept full transformability after being stored for at least one month at -80ºC [14]. For these reasons, we expect that this protocol can be easily adapted to other marine strains such as P. nigrifaciens strain 217.
Below is the adapted protocol [14] that describes the steps that should be followed to transform the plasmids in P.nigrifaciens:
1.Cultivate the Pseudoalteromonas strain overnight at 24ºC.
2.Place overnight cultures on ice and wash 2ml aliquots 3 times with 1 ml of an ice-cold modified sucrose buffer, containing 272 mM sucrose, 1 mM MgCl2,
7 mM K2HPO4 adjusted to pH 7.5 and sterilized by autoclaving.
3.Immediately before use, add 15% glycerol to the buffer to avoid cell lysis.
4.After 3 washing steps, concentrate electrocompetent cells in 50uL with the same buffer and place the cells in an ice-cold 1 mm electroporation cuvette, mix them with 200 ng of plasmid DNA and directly electroporate them with a Gene Pulser II + Pulse Controller Plus (Biorad) set at 1.5 kV, 200 resistance and 25 F capacitance.
5.Immediately after electroporation, add 1 ml of Pseudomonas bathycetes (PB) adapted medium and allow cells to recover for 4h at 24ºC.
6.Plate cell suspensions on kanamycin plates and incubate for 2-3 days at 20ºC.
