Although cloud seeding can be done using pure INPs, a bacterial ghost is constructed, which will be more efficient in freezing supercooled water. On top of that is the downstream processing of the pure protein more complex in comparison to that of the bacterial ghost. A bacterial ghost consists of an
E. coli that expresses INPs on its outer membrane, which the INPs perforate at a certain point. This causes all the cell organelles and cytoplasm to be expelled from the bacteria which results in an empty cell envelope. The gene responsible for this phenomenon is the lysis E gene. The protein E expressed by this gene makes tunnels in the membrane of the bacteria causing it to expel its cytoplasmatic contents and kill the bacteria in the process. This also creates a unique opportunity because the bacterial ghost construct will no longer be considered to be GMO.
To achieve this, a plasmid is designed that contains both
InaZ (INP gene) and protein E gene on the same backbone. This plasmid is constructed using multiple techniques like PCR, CPEC and BioBricking. The plasmid is then used to transform
E. coli cells. After transformation the bacteria are grown through fermentation. The INP gene has a constitutive promotor which means that the INP expression starts immediately, without the need of an inducer. The protein E gene is preceded by an inducible promotor. Cell lysis is induced when the stationary phase of growth starts. This is determined using optical density (OD) coupled with cell dry weight (CDW) or colony-forming unit (CFU).
After fermentation, the batch of cells must undergo further downstream processing. First of all, the biomass of the medium is separated by means of Tangential Flow Filtration (TFF) (Langemann et al., 2010) or centrifugation (Amara, Salem-Bekhit, & Alanazi, 2013); (Rabea et al., 2018). In order to guarantee that the product does not contain GMO’s, all remaining viable cells and DNA have to be destroyed which can be done by using β-propiolactone (Amara et al., 2013). Subsequently, the biomass is separated from other cell material and chemicals by applying diafiltration. Finally, the cells are washed several times with sterile water. The bacterial ghost construct will then be lyophilized to be compatible with pneumatic flares as further discussed in the part about dispersion.
To test whether or not our E-lysis was efficient, the CFU of the samples taken before and after the fermentation are determined. The efficiency will be highest when all bacteria are lysate, this will result in very few or no CFUs after lysis.
The efficiency of the ice nucleation activity of the bacterial ghost construct can be tested by performing a droplet freezing essay. In this assay the bacterial ghosts are added to a number of water droplets. By repeating the test with a series of temperatures and looking at which temperature most droplets freeze, the freezing temperature of the droplets with ice nuclei can be determined.
TARU – MK1, a pneumatic flare. Source: Cloud Seeding Technologies 
