Aminoglycoside phosphotransferase gene (APHVII) from
treptomyces hygroscopicus can be used as an optional marker for
Chlamydomonas reinhardtii to establish hygromycin resistance. Since our chassis organism is
Chlamydomonas reinhardtii, we optimized its codons and added introns for it. From registry, we found that our APHVII gene associated with the gene from iGEM19_ Humboldt_Berlin team's is very similar (
BBa_K2984012 ) by BLAST method. After comparison, the DNA sequence we found is slightly shorter, the matching region is only different in two base pairs, and the protein sequence is totally identical(
Figure 1). But we found that the results and data provided by iGEM19_Humbolt_Berlin are very few while we have done more verification on this gene.
Figure 1. Our comparison with the APHVII Sequence of iGEM19_Humboldt_Berlin. The upper line is BBa_ K2984012 and the lower line is BBa_ K4335003.
In order to verify that our assembly is successful, we designed two primers, namely
HgR-F and
HgR-R for PCR amplification verification. And we amplified about 600 bp of fragments, which indicates that we successfully inserted HgR into our vector(
Figure 2).
Figure 2. Single colony PCR verification of Hyg resistance gene.
We transferred the vector we constructed into Chlamydomonas reinhardtii by electrotransformation. After screening on the solid plate, some positive clones containing hygromycin resistance grew out (Figure 3). At the same time, we performed PCR verification on the single colonies of screened algae (Figure 4). In order to reduce the interference of false positive, multiple screening was carried out and then eight clones in the plate were randomly selected from 96-well sterile plate for re-culturing (Figure. 5A). After a few days, the number of surviving clones (shown as green liquid) was counted (Figure. 5B). Subsequently, to amplify the positive clones obtained, three successful random clones were transferred to a 24 well 24-well plate for culturing cell (Figure 5C). After the above process, Chlamydomonas reinhardtii could still make it to survive, which proved the success of our transformation.
Figure 3. Selection and genetic editing efficiency of thaumatin-resistant colonies after transformation.
Figure 4. Gel run of samples from colony PCR. HgR: Hyg resistance gene.
Figure 5. Statistics of multiple screening of Chlamydomonas reinhardtii strain after transformation of plasmid empty vector.
(A) The positive result of the control group is the growth condition of wild type algae in TAP medium without hygromycin screening pressure, and the negative result is the growth condition of wild type algae in TAP medium with 25μg/mL hygromycin screening pressure. The green color of the algal liquid indicates that it can grow normally. The slightly light color or transparency of the algal liquid indicates that the cells are screened.
(B) Statistics of positive data of 8 clones in a 96 well plate;
(C) Three clones randomly selected from 96 well plates were screened for the third time on 24 well sterile cell culture plates. All holes contain 1mL TAP+25μg/mL Hyg medium.
mCherry is a red fluorescent protein derived from the fluorescent reporter gene of mushroom coral that can be used for biological applications. Since we have optimized its codon and added introns for our chassis organism Chlamydomonas reinhardtii, mCherry can be expressed more efficiently in Chlamydomonas reinhardtii.
In registry, we found that the sequence of our mCherry largely resembled the sequence of iGEM16_USP_UNIFESP-Brazil's(
BBa_K2136016) by BLAST method. After sequence alignment
(Figure 6), it is found that there is only one base pair difference between the sequence of iGEM16_USP_UNIFESP-Brazil's mCherry and the coding sequence of ours. The protein sequence from both sides is the same while the difference is that we add introns to it.
Figure 6. The blue strip at the top is BBa_ K2136016 (iGEM16_USP_UNIFESP Brazil), the red band in the middle is the coding sequence of our mCherry, and the dotted line represents the intron sequence
In plasmid
(Cas9+gRNA+mCherry+ ChlamyHgR) , mCherry is used as its reporter gene, PCrRBCS2 is used as the promoter, and TCrRBCS2 is used as the terminator to achieve the expression of mCherry gene. To verify that our construction is successful, we designed
mCherry-F and
mCherry-R primers for PCR gel running verification (
Figure 7). Subsequently, we screened single colonies after electrotransformation, performed algal colony PCR and observed fluorescence under the fluorescence microscope (
Figure 8 and 9).
Figure 7. PCR validation of mCherry gene fragments using transformed monoclonal Escherichia coli cells.
Figure 8. PCR validation of mCherry gene fragments using transformed monoclonal algal cells.
Figure 9. Images of WT Chlamydomonas reinhardtii and positive clones after transformation at 20x magnification. WT: wild type; DIC: bright field. 60ms exposure time; Chlorophyll: EX:Form 625nm to 650nm, 10ms exposure time; mCherry: EX:Form 515nm to 555nm, 300ms exposure time; StayGold: EX:Form 465nm to 495nm, 300ms exposure time.
Based on the above results, we can determine that mCherry gene, which has been through codon optimization, can be successfully expressed in Chlamydomonas reinhardtii and eRED fluorescence was observed.
1. Bläsing, L., Goebel, G., Flötzinger, U., Berthold, A. & Kröner-Herwig, B. Hypersensitivity to sound in tinnitus patients: An analysis of a construct based on questionnaire and audiological data. International Journal of Audiology 49, 518–526 (2010).
2. Greiner, A. et al. Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9. The Plant Cell29, 2498–2518 (2017).
