After species confirmation of Debaryomyces hansenii by ITS sequencing, wild-type salt tolerance testing was the first step taken to determine the ability of the yeast to grow in high concentrations of salt (sodium chloride).
● A single colony was picked and added to SDA media with different concentrations of Sodium Chloride (26g/L-50g/L-100g/L-150g/L) and left overnight at 25 °C - 200rpm.
● The next day the culture was diluted with ratio 1:5 using SDA media. Then, OD600 was measured to find the optimum salt concentration for growth.
To determine the amount of salts that could be accumulated in yeast naturally, a 50g/L NaCl SDA media which was found to be the most optimum concentration for debaryomyces hansenii growth was used.
● A single colony was inoculated and left overnight shaking at 200rpm - 25 °C.
● The next day the culture was diluted 1:10 with deionized water and the total dissolved salts were measured and recorded after that a further suspension from the overnight culture was taken and diluted 1:10 using SDA (50g/L NaCl) then we further took 1mL added it to 9mL deionized water and measured the TDS again at different intervals. (15, 30, 45, 60,75, 90, 105, and 120 mins)
At first we aimed to identify binding affinity of different metal binding proteins from iGEM registry using in-vitro expression system. . Taking into consideration the promoter and ribosomal binding site strength, the circuit was designed so that coding sequence is under the control of constitutive promoter family member "BBa_J23102” and strong RBS “BBa_B0034” with a bidirectional terminator at the end “BBa_B0011”.
Using cell-free kit from Arbor bioscience, Four metal-binding proteins ( CutA1, CSP1, CRS5 , bacterioferritin) were expressed and tested for their affinity to bind sodium and chloride ions.
Different time intervals were tested to find the best production time and largest protein quantity.
To measure protein concentration, Expasy Protparam tool was used to identify the theoretical coefficient and then Proteins’ concentrations were calculated using
Beer’s law: A=εBC*
Proteins binding affinity was detected using TDS meter, knowing that TDS readings indicate organic and non-organic ions regardless of macromolecules such as proteins, lipids or sugar.
● A 70g/L sodium chloride solution (Twice sea water salinity) was used. different pHs, ranging from 3 to 9 and different temperatures (4 °C,25 °C,37 °C,42 °C and 68 °C) were tested to check for the best binding conditions.
Testing the binding was done by diluting the NaCl solution containing 9 ul of protein solution with dilution ratio 1:10, as TDS sensitivity range we have ranged in 9990 ppm only.
From the results of the modeling, we compared the affinity of bacterioferritin to bind sodium and GST tagged bacterioferritin in room temperature, other temperatures tested at were 4 C,25 C,37 C,42 C, and 68 C; the last temperature was chosen according to the result of stability curves produced from modeling of CSP1, Bacterioferritin.