Transformation of Deinococcus geothermalis

 Introduction: 

    In the previous week, four plates were inoculated with two different cultures of D. geothermalis in order to evaluate transformation efficiency of Prad. This plasmid from E. coli can be useful for future transformation experiments that would place selective pressures on the CRISPR-cas system of D. geothermalis, which is very detailed and described in comparison to others in the genus such as D. aquaticus. The two cultures were of one control group that underwent the transformation process and but was not exposed to the actual plasmid, whereas the experimental was. The plates that were grown for those 72 hours were evaluated for transformation and possible contamination, and another attempt was made using the same protocol after more precautionary measures were taken. Other plates were also inoculated so that the potential source of contamination in the previous plates could be narrowed down. 

Methods:

    The plates from the previous transformation showed no sign of growth over 72 hours and were discarded. 

      A new transformation attempt was completed using the second competent cell tube that was made the week prior, this time allowing for 16 hours of growth at 40 degrees Celsius before being plated on either antibiotics or an standard TGY. This week control group was utilized that was inoculated from the competent cell sample and underwent the transformation protocol, but was not exposed to plasmid. The two cultures were gram stained and confirmed as D. geothermalis before going to plate, but the control group was a milky white culture as opposed to the standard orange color expected of the this species. The experimental group did not show signs of significant growth or any coloration visible to the naked eye. Growth of the plates was evaluated at 24. 48, and 72 hours after incubation at 37 degrees Celsius. 

        Two of the plates were dosed with chloramphenicol (3 ug/ml) prior to pouring and the other two were standard TGY. Both cultures were grown on each type of plate to confirm results. After 72 hours, which aligns with the standard incubation time of D. geothermalis, significant growth was seen on all but one plate, which was the transformed cells on the chloramphenicol plate. The other three plates were gram stained to evaluate for identity of the cells and possible contamination. 

    Once contamination was confirmed, the source of the contamination along the process was attempted to be clarified. Both competent cell tubes (number 1 and 2, which were individually used for both attempts at transformation) were four way streaked onto standard TGY plates, as well as the original broth culture that was used to make the competent cell tubes, just in case the contamination was inevitable and the sources of the cells were actually tainted as opposed to just poor aseptic technique during plate inoculations. These plates were allowed to grow for 72 hours at 37 degrees Celsius. The broth cultures that were used in the first plating, the second attempt at transformation, were used to inoculate new plates in the hood instead of in the general area and also allowed to grow for 72 hours at 37 degrees Celsius. 

Results:

     The first set of the plates in the beginning of the week after 16 hours of growth in the experimental and control tubes all showed varied levels of contamination with staphylococcus. The plate with chloramphenicol and cells exposed to Prad1 did not experience growth so no gram stain could be accomplished. 

    Nearly each plate had colonies of different colors, which is reflected in the gram stains as well. 

Figure 1: This plate had cells that were part of the control group (not exposed to plasmid during transformation process) and were found on the plate dosed with chloramphenicol. There are gram positive tetrads that indicate D. geothermalis, but also smaller gram negative cells. 

Figure 2: This plate was control cells on a plate without chloramphenicol, but was one of the visibly orange colonies on the plate as opposed to other white colonies also seen on the same plate. There is numerous gram positive tetrads indicating D. geothermalis, but there is still evidence of small cocci shaped cells in the background. 

Figure 3: This plate had cells that were part of the control, and standard TGY with no chloramphenicol. This was a white colony that was seen on the plate and evaluated separately from orange colonies seen on the plate. There is a few gram positive D. geothermalis cells, but an overwhelming abundance of what appears to be staphylococcus contamination. 

Figure 4: These were the three plates gram stained after 72 hours of growth, the top plate producing gram stains in figures 2 and 3, the middle plating for figure 5 and the bottom plate figure 1.  

Figure 5: This plate was cells potentially transformed, exposed to the plasmid in the transformation process but only grew on the TGY plate not dosed with chloramphenicol. There is distinct evidence, again, of both gram positive and gram negative cells indicating D. geothermalis with contamination of likely staphylococcus. 

Discussion: 

    The results from this week obviously show issues in the plating technique during this process. The question was really whether or not the competent cells, in either tube, are also contaminated, dooming the transformation process completely, or if it was a step along the way that caused this problem. That being said, the plates inoculated to confirm the correct identity and lack of contamination in the competent cells tubes will reveal which step was the culprit, but it is assumed that it was during plating, especially since it was not originally done in the sterile hood. Hopefully by carrying out these inoculations for transformation in the hood will prevent these issues in the future attempts. There is a chance since the broth cultures post transformation and prior to plate gram stained clean that the newly inoculated plates will show evidence of a successful transformation. I believe it will also be more beneficial to grow these broth cultures at a temperature more suited for D. geothermalis, such as 40 to 45 degrees Celsius. It is also curious as to why the original broth culture used to make the competent cells was bright orange but grown at 45 degrees Celsius and the broth culture post transformation grown at a lower temperature is somehow much more visibly white. It may indicate some sort of interruption in the carotenoid pathway of the cells depending on the growth temperature. This may be something to look into in the future as well. If these cells are not transformed with these plates, it may be beneficial to make new competent cell tubes with a different ratio of chemical agents that was found in a separate paper, that has small adjustments to the protocol that Jonathan uses for D. radiodurans that we are now currently using.