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. Our improved part is registered here
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 total number of colonies we discovered that the number of colonies was much higher for the improved plasmid version, regardless of the amount of plasmid at the time of electroporation. The colonies of the samples using the standard usage resulted in a total amount of 175, whereas the improved plasmid version produced 665 colonies (Fig. 2). Comparing the plasmid mass we discovered that the amount of colonies does not strictly correlate to the amount of DNA used during the electroporation (Fig. 3). 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 DNA mass. Yet, further tests should be made to examine the exact effect of DNA mass 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 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. 4. 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.reinahrdtii colonies capable of growing TAP-paromomycin-agar plates. 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 transfer RNAs 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 µM the algae need a certain minimum amount of protein expression to survive the plating. 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 and 600 . The majority our electroporations were performed within this range and and its effect on the number of counted colonies should be neglectable.
This experiments clearly indicate 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 rimosusaphVIII 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.