Part:BBa_K4335003

ChlamyHgR

Berthold et al. (2002)([1]) found that the aminoglycoside phosphotransferase gene (aphVII) from Streptomyces hygroscopicus works as a selectable marker for C. reinhardtii establishing a hygromycin resistance.

BBa_K4335003 is a resistance gene optimized for Chlamydomonas reinhardtii based on the original gene. We inserted a 145bp intron into the original gene to facilitate its expression in Chlamydomonas reinhardtii.

Introns are ubiquitous in eukaryotes and are an important feature that distinguishes them from prokaryotes. In higher organisms, introns have been reported to regulate expression at multiple levels. The main function of introns is to generate different exon combinations by alternative splicing and then translate different proteins, which improves the complexity of the proteome. [2]

Validation

We found from the registry page that BBa_K2984012 from iGEM19_Humboldt_Berlin's team was very similar, so we compared BBa_K4335003 with BBa_K2984012 using snapgene (Figure 1)

Figure 1 Comparison of BBa_K4335003 and BBa_K2984012 DNA sequences

The sequence difference between the two is only two bases. And the sequence of the protein expressed is exactly the same.

Usage

We inserted ChlamyHgR into plasmids pTX2038 and pTX2040 as marker genes for transformation screening.

Result

Plasmid construction

To verify our successful assembly, we designed two primers, Hgy-F and Hgy-R, for PCR verification. The primer location, primer sequence and gel map are shown in the following figure:

Figure 2.The primers：Hgy-F and Hgy-R

Figure 3.The length of the amplified sequence was 606bp；M is DNA Marker.

The determination of optimal screening concentration

According to the specificity of the codon of Chlamydomonas reinharditii, our team optimized and designed a new part for the ChlamyHgR (Part:BBa_K4335003) based on the existing part, and the new part acted as the selection standard for transforming positive clones. However, the inhibition effect of hygromycin varies with species. Therefore, we designed and performed experiments to determine the minimum inhibitory concentration of Hyg on the growth of Chlamydomonas reinhardtii in solid medium.

Figure 4. The wild type Chlamydomonas reinharditii CC-503 (2 × 10⁸ cells) cells were placed on TAP solid medium supplemented with different concentrations of Hyg (10, 20 and 25 µg/mL), and the growth of Chlamydomonas reinhardtii on the agar plate was continuously observed. The pictures displayed correspond to a 2-week old culture. The experiment was repeated in three copies.

After two weeks of incubation, we observed an inverse relationship between the number of colonies growing in the culture dish and the concentration of Hyg (Figure 4). Only in the plates with a concentration of 25 µg/mL Hyg we consistently observed inhibition of cell growth. Therefore, we decided to use this concentration in TAP media to carry out the experiments described below.

The procedure of transformation

We chose to use the method of electrotransformation to transform Chlamydomonas reinhardtii. In order to improve the efficiency of electrictransformation, we used electroporation buffer (ME Suc): Use convert reagent with MAX efficiency ^TM(Therfisher, # A24229) to carry out the experiment.

1×10⁸ cells of algal solution and 2 to 4μg of linearized plasmids were used in each electrotransformation. The linearized pTX2038 and pTX2040 vectors were respectively electroporated into Chlamydomonas reinhardtii strains at 635V, 31μF, and 800Ω. The cells were allowed to recover for 14-16 hours under the condition of continuous light and vibration treatment. Single colonies were separated on AGAR plates containing 25μg/mL Hygromycin. For detailed information, you can refer to our working procedures (Figure5).

Figure 5. Summary of the working procedures to produce mutants by using genetically encoded Cas9.

After 7 days, the resistant colonies which grew vigorously were obtained in the experimental group (Figure 6A), while no algae grew in the negative control group. The number of monoclone which had been successfully transferred into pTX2038 and pTX2040 in the plate was counted respectively and their transformation efficiencies was also calculated respectively (Figure 6B), after which process the editing efficiency of 1.9-2.1×10^-6 was achieved, and a relatively effective transformation system of Chlamydomonas reinhardtii was established.

Figure 6. Selection and genetic editing efficiency of thaumatin-resistant colonies after transformation. (A) Growth of Chlamydomonas reinhardtii in the plates after electrotransformation (7 days). Negative control: no plasmid was added. All dishes shown in the figure contain TAP medium supplemented with 25 µg/mL of Hyg, except for the positive control of the wild-type Chlamydomonas reinhardtii strain. (B) Statistics of positive clones after transformation ang the frequency of transformation.

Molecular test

To verify whether the transfer of the vector into 503 cells of Chlamydomonas reinhardtii at the molecular level is successful or not, we used the colony PCR method to ensure full integration of the vector.

We extracted the DNA from the above amplified algal solution after successful transformation by heating lysis at 95℃, and designed primers with a length of about 600bp from four mutually owned fragments of vector, namely Cas9 protein, Hyg resistance gene, mCherry reporter gene and StayGold reporter gene. The Hyg resistance gene and Cas9 protein were shared by the two vectors, and the reporter genes mCherry and StayGold were owned by vectors pTX2038 and pTX2040, respectively. For the PCR amplification reaction on the DNA of the transformed single algal colony, we also set up a linear plasmid of the vector as a positive control group.

Figure 7. Gel run of samples from colony PCR. The amplification of single algal colony. (A) Sequence comparison of Cas9, HgR, and mCherry fragments in pTX2038. (B) Sequence alignment of Cas9, HgR, and StayGold fragments in pTX2040. .M: 2000bp DNA marker; NC: wildtype; PC: Linear plasmid. 1-3 indicate the different transformants selected.

The monoalgal colonies of pTX2038 and pTX2040 have been transferred. The brightness of PCR products of Hyg resistance gene, mCherry reporter gene, Cas9 protein and StayGold reporter gene (Figure 13A and 13B) fragments were all consistent with the DNA bands of the positive control group as well as being matched with the position of DNA Marker, which indicates that the fragmented PCR products transferred into the vector were in a high concentration and normal expression state. In sum, the effectiveness of transformation could be testified.

Reference

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 782
  Illegal NgoMIV site found at 890
  
• 1000
  COMPATIBLE WITH RFC[1000]