Introduction
Paromomycin belongs to a group of aminoglycoside antibiotics such as neomycin or dibekacin. These aminoglycosides are capable of inhibiting the eukaryotic translation, by binding within the large and small subunit of the 80S ribosome. This property allows paromomycin to be used as selection marker for C. reinhardtii. For the selection process to work, one must consider a way to implement a paromomycin resistance in C. reinhardtii. The bacteria Stretpomyces rimous carries the aminoglycoside 3’-phosphotransferase encoded in the so called aphVIII gene. This enzyme catalyses the transfer of the gamma-phosphate of ATP to the hydroxyl group in 3’ position of the paromomycin molecule and allows the carrier of the gene to develop a resistance to paromomycin (Sizova et al. 2001). We used this resistance as a screening method for most of our transformations. During our research we discovered, that this resistance gene was already contributed to the iGem Registry. We wanted to improve this part by changing the codon usage and see if this improvement would end up in a higher expression of the aminoglycoside 3’-phosphotransferase and therefore in a better resistance to paramomycin.
Methods
To see if the expression of the aminoglycoside 3’-phosphotransferase was increased, we performed several electroporations to transform C. reinhardtii with the paromomycin resistance. We used the C.reinhardtii strain UVM 4 since it is a strain designed to express transgene constructs (Neupert et al. 2009). We compared the two paromomycin constructs with standard and improved codon usage starting with 0,5 µg DNA per electroporation sample and ascended with 0,5 µg steps up to 2 µg. For each construct and DNA mass we did three electroporations. The electroporation electrical resistance was measured for each sample. After resuspension and one day recovery in TAP medium, all samples were plated on TAP-agar plates containing a paromomycin concentration of 10 µM. After two weeks of growth, colonies corresponding to each sample were counted. Each colony of C.reinhardtii represents a successful transformation of the resistance and indicates the expression of the aminoglycoside 3’-phosphotransferase. By counting the amount of colonies on the plates, we could determine which construct and at which DNA mass at the time of transformation worked best.
Results
Counting the colonies we discovered that the number of colonies was much higher for the improved plasmid version for every amount of plasmid. The colonies of the samples using the standard usage resulted in a total amount of 175 whereas the improved plasmid version produced 665 colonies. Comparing the plasmid mass we discovered that a higher amount does not necessarily result in a larger number of colonies. Shown in figure the mean number of colonies using the standard-plasmid is higher for 1 µg of DNA then for 1,5 µg. Same can be observed when taking the improved Version into account. Here the amount of colonies for 0,5 µg ist higher than for 1 and 1,5 µg. One explanation for the variable amount of colonies might be the inconsistency of the electroporation resistance. To see how the electroporation process affected the number of colonies their quantity was compared with the corresponding resistance. The left hand figures depicts that the approach with 1,5 µg standard plasmid was executed with a robust resistance around 570 for all 3 samples. The 1 µg approach of the same plasmid shows a variable resistance but delivered more positive colonies. In the 2 µg standard plasmid approach the resistance of the first sample dropped to 457 but the same amount of colonies as in sample 1 of the 1,5 µg standard approach were counted. With further comparison of these to approaches it can be seen, that the third samples in the 1,5 µg and 2 µg sets showed similar resistance but the third sample of second approach resulted in a much higher colonie number. The data behaves similar for the improved plasmid. The second sample of the first set and the third sample of the third were carried out with a resistance around 610 but for the third sample almost 34 more colonies were counted.
Discussion
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