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We found an unpublished version with improved codon usage designed by Irina Sizova.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.
Discussion
The experiments showed clearly, that the improved codon usage resulted in larger number of C.reinhardtii paromomycin-resistant colonies. This proves that the changed codon usage promotes a higher expression of the aminoglycoside 3’-phosphotransferase.
Nevertheless, there are many factors that can influence the expression of the resistance gene. During the electroporation process, transgene DNA is inserted randomly inside the genome. Depending on the regulation imposed on that genomic region, DNA transcription frequency varies, since it changes with the necessity of the genes usually coded in that same region. This effect could be bypassed through targeted insertions, something we tried with our CRIPSR/Cas9 experiments(Link zur Seite). Additionally it has to be considered that for high expression, the availability of tRNAs is decisive. Each individual organism features a different set of tRNAs capable of matching certain types of mRNA codons. If the transgene DNA is made of codon where the organism is lacking compatible tRNA’s, the translation might be slowed down. This is what we avoided by codon optimizing the resistance gene. Since all our algae clones were exposed to the same paromomycin concentration of 10 µg/ml the algae need a certain minimum amount of protein expression to survive. If the DNA is inserted inside a locus that features lower expression rates, the expression might be not high enough to withstand the pressure of selection. With the improved usage, a locus with similar expression rate might be above that minimal amount and the algae can survive despite the locus restraints.
Besides the structure of the genome, expression of transgene DNA can be influenced by be the design of the transgene construct itself. In this case the same construct design was used for both versions of the codon usage. The constructs consist of the AR promoter, an Intron fused to the beginning of the paromomycin resistance gene and the RbcS2 terminator at the end. In addition to genomic background and transgenic design, the setup determines the outcome of an experiment. One factor is the fitness of the cells. In preparation of our electroporation, we decided to leave out the heat shock and reduced the centrifugation speed to 1.250 rpm. Each pipetting step was done with high precaution to reduce damage of the cells. A stable resistance is crucial for a successful transformation. If the resistance is too high, the electrical pulses might not be strong enough to pour the membrane and transfer the DNA. On the other hand, a low resistance enables stronger currents, which can lead to higher cell death rate. The resistance should vary between 400 - 600 Ω. The majority of our electroporations were performed within this range and and its effect on the number of counted colonies should be neglectable.
These experiments clearly indicate enhanced functionality of our improved construct. For future transformations this antibiotic resistance gene can be used as a powerful screening tool boosting up the number of positive clones.
We hope that our contribution helps future iGEM Teams working with Chlamy to increase their positive results.
Sources
- Sizova, I., Fuhrmann, M., & Hegemann, P. (2001). A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene, 277(1-2), 221-229.
- Neupert, J., Karcher, D., & Bock, R. (2009). Generation of Chlamydomonas strains that efficiently express nuclear transgenes. The Plant Journal, 57(6), 1140-1150.
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