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Part:BBa_K4335022
Rbcs2 Promotor+ChlamyHgR+Rbcs2 Term
BBa K4335022 consists of a promoter, a resistance gene, and a terminator. The resistance gene APHVII[BBa_K4335003] was optimized for Chlamydomonas reinhardtii on the basis of the progene. We inserted a 145 BP intron into the progene to facilitate its expression in Chlamydomonas reinhardtii.
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:![](https://static.igem.wiki/teams/4335/wiki/zhhyg4.jpg)
Figure 2.The primers:Hgy-F and Hgy-R
![](https://static.igem.wiki/teams/4335/wiki/zhhyg10.png)
![](https://static.igem.wiki/teams/4335/wiki/zhjt5.jpeg)
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.![](https://static.igem.wiki/teams/4335/wiki/zhhyg3.png)
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.
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).
![](https://static.igem.wiki/teams/4335/wiki/zhhyz2.png)
Figure 5. Summary of the working procedures to produce mutants by using genetically encoded Cas9.
![](https://static.igem.wiki/teams/4335/wiki/zhhyg1.png)
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.
![](https://static.igem.wiki/teams/4335/wiki/zhjt3.jpg)
Figure 7. Gel run of samples from colony PCR. The amplification of single algal colony.Sequence comparison of HgR fragments in pTX2038 and pTX2040. M: 2000bp DNA marker; NC: wildtype; PC: Linear plasmid. 1-3 indicate the different transformants selected.
Reference
[1]Berthold, P., Schmitt, R., & Mages, W. (2002). An engineered Streptomyces hygroscopicus aph 7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. Protist, 153(4), 401-412[2]Parenteau, J. et al. Introns are mediators of cell response to starvation. Nature 565, 612–617 (2019). Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1236
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1121
- 1000COMPATIBLE WITH RFC[1000]
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