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. The bacteria Streptomyces rimosus carries the aminoglycoside 3’-phosphotransferase encoded in the so-called aphVIII gene. This enzyme inhibits paromomycin by transferring 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. The aphVIII gene is a commonly used transformation marker in green algae and established for the use in C. reinhardtii (Sizova et al. 2001) and was submitted to the registry last year as BBa_K2703008.

However, despite its broad distribution, the sequence is not fully optimized to the codon usage of C. reinhardtii. As we planned to use the paromomycin marker in our C. reinhardtii transformations we set out to use the most efficient part.

We found an unpublished version with improved codon usage designed by Irina Sizova. In order to evaluate if this improvement would end up in higher expression of the aminoglycoside 3’-phosphotransferase and therefore in a better resistance to paromomycin we recloned this part to meet the MoClo syntax and can be included in our parts collection. Our new part with improved codon usage is registered under BBa_K2984006.

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 UVM4 since it is a strain designed to express transgene constructs (Neupert et al. 2009). We compared the old standard aphVIII gene with the improved codon usage starting with 0.5 µg plasmid DNA per electroporation and ascended with 0.5 µg steps up to 2.0 µg. For each construct and DNA concentration we did three electroporations. The 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 µg/ml. See our protocol section for details. After two weeks of growth, paromomycin-resistant colonies were counted. Each colony of C. reinhardtii represents a successful transformation of the resistance gene 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 plasmid concentration at the time of transformation worked best.

Results

Counting the total number of colonies we discovered that the number of colonies was much higher for the improved aphVIII version. The old aphVIII resulted in a total of 175 colonies, whereas the improved version produced 665 colonies (Fig. 3). Comparing the plasmid concentration we discovered that the amount of colonies does not strictly correlate to the amount of DNA used during the electroporation (Fig. 4). For the paromomycin resistance with standard codon usage we can see that the number of colonies at 1.5 µg is smaller than expected. Similarly, the amount of colonies for the improved resistance at a DNA mass of 0.5 µg is much higher than expected. The other results seem to show a tendency of increasing colony numbers with more plasmid DNA. Yet, further tests should be made to examine the exact effect of plasmid concentration during transformation for these parts. As can be seen on Fig 3., the mean number of colonies using the standard-plasmid is higher for 1 µg of DNA then for 1.5 µg. The same can be observed when taking the improved version into account. Here the amount of colonies for 0.5 µg ist higher than for 1.0 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. Fig. 5. depicts that the set with 1.5 µg standard plasmid was executed with a robust resistance around 570 for all 3 samples. The 1 µg set of the same plasmid shows a variable resistance but delivered more colonies. In the 2 µg standard plasmid set 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 set were counted. With further comparison of these to sets 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 the second set resulted in a much higher colony 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.