Karolina Jurkiewicz
Cebic Fellowship 2005,Princeton University
Prof. Francois Morel Lab, Geosciences Dept.
Supervision of PhD. Candidate Heawon Park
My work was focused on microscopic organisms –diatoms, mostly concentrating on the role of Cadmium in their cells. Carbonic anhydrases are enzymes in diatom’s periplasm or cytoplasm responsible for equilibrium of HCO3 and CO2 and they supply inorganic carbons for photosynthesis. Carbonic anhydrases require metal Zinc to function. In some marine organisms like Thalassiosira weissflogii CA can use metals like cadmium, cobalt and zinc to increase the level of CO2 inside the cells. These metals can substitute each other to maintain cells’ optimal growth rate. (In particular, the addition of Cd was known to enhance the growth rate of some marine phytoplankton). The goal of my research was to culture the colonies of T. pseudonana in different concentrations of Cadmium and Zinc and to see how it effects their growth rate as well as find the concentration of Zn that limits the growth rate. I also, would like to see how similar is TP to TW in the function of different metals in photosynthesis. The reason why the Tp colony is very interesting is the fact that their genome was sequenced and there was found the gene CDCA similar to that of T. weissflogii, however the functional expression of the gene is unknown.
Second goal was to present how different concentrations of Zn and Cd can influence the expression of the gene CDCA (which contains Cadmium) and also culture and analyze many other members of this family searching for that gene.
I had conducted three experiments involving T. pseudonana colony. It was six bottles with seawater and enriched by various nutrients (NaH2PO4, NaSiO2, NaNO3), metals and minerals. Six different concentrations: 1Zn’, 2Zn’, 3Zn’, 1Zn’+10Cd’, 2Zn’+10Cd’, and 3Zn +10Cd. 1Zn’ corresponds to ~ total concentration of Zn = 100uM x 16.14ul/250mL, 100uM EDTA=6456*10-9. Since CAs in diatoms can utilize both Zinc and Cadmium for the process of photosynthesis, my hypothesis was that 3Zn +10cd would be the best for their growth. Two of the three experiments did not show the results I expected. Probably the contamination occurred during the procedure or the starter cells were dead. The last experiment showed that more cadmium and zinc there is, greater the growth rate. Also, despite contamination in two first colonies, I found the maximum growth rate of T. pseudonana. It is about 1.5/day. The reduced growth rate was about 50% lesser then maximum.
· These results show possible contamination in the colony.3Zn+10Cd are the greatest, but there is no consistence in the rest of the concentrations
· My hypothesis was proved by this experiment. Growth rates are reasonable +we can also see clearly that presence of Cd in any concentration of Zn makes the colony growth faster than lack of cadmium in colonies: 1Zn, 2Zn, 3Zn.3zn+10Cd is the greatest.
After culturing three T. pseudonana cultures as well as many others, like: Cm and Pt my goal was to see their CDCA gene expression.
I was trying many different members of diatoms to see their similarity in the expression of CDCA gene. Results varied since I used a lot of different primers. One of the best results I’ve got was probably RNA expression of CdCa. Both Tp and To colony was cultured in different concentrations. To was growing in 0.5 Zn+6Cd and Tp was growing in 2Zn+10Cd. In above picture I used primers-18 S (F+RC) and CdCa (F+RC). The PCR was programmed for temperatures: 94 C-30 sec., 54 C –30sec., 72 C-1 min.
· In the first four samples 18 S was used as a primer. The two first bands represent TW (duplicate). The third one is TP and forth TO. The next four bands represent TW (duplicate), TP and TO. Clearly, the gene is expressed, TO and TP probably at length 400 bp and TW at 1000 bp. This experiment gave me the idea that there is CdCa gene expression in different members of diatoms.
Next, my goal was to see the expression of CdCa in TP cells (from different concentrations of Zn and Cd) and to see if this could have some effects on their gene expression. After the RNA purification, I did RNA quantification and the intensity and concentration of various TP samples were too low to make any cDNA. There were many reasons why I did not get enough RNA:
· Starter or collected cells were dead
· It was not enough RNA (cells)
· Contamination during procedure (extraction RNA or purification, it is a very clean work)
· This particular picture represents the RNA of: Ladder, 1 Zn’+10Cd’TP, 1 Zn’TP,2 Zn’+10Cd’TP,2Zn’TP,3Zn’+10Cd’TP,3Zn’TP ,and Phaedactylum Tricornutum,D.Brightwelli, Chaetoceros Calastrant and DNA of TP. The picture clearly indicates that there are no RNA bands. The unclear, bright places indicate probably DNA or contamination.
During my summer research I learned many different methods and techniques. Culturing as well as molecular biology skills:
Methods and Techniques on diatoms:
-Preparation of the Medium
-Incubation of the cells
-Counting cells
-Extraction of the DNA, RNA
-Quantification of RNA
-Making CDNA
-PCR using both RNA and DNA