Part:BBa_K4904002

BD-CRY2-N489-GFP

Composite Part - BBa_K4904002 (BD-CRY2-N489-GFP)

Construction Design

The original plasmid must be reconstituted and rebuilt in the new blue light control switch. The original plasmid structure of pbridge-BD (BBa_K4904004) was modified. GFP (BBa_E0040) (Green fluorescent protein) and UAS (BBa_K4904000) (Upstream activating sequence) gene fragments were inserted into pbridge-BD and added into Escherichia coli, and then the recombinant plasmid was extracted [1-2].

Figure 1. Map of BD-CRY2-N489-GFP

Engineering Principle

The blue-light-activated CRY2 protein forms photobodies through phase separation to recruit the m6A "encoder" complex, which regulates transcriptome methylation. m6A methylation changes mRNA's degradation rate, the biological clock's core component, thus affecting the circadian clock's light response [3].

Figure 2. Gene Map of CRY2-N489

Green fluorescent protein (GFP) is a β-barrel-shaped protein composed of 238 amino acids with a molecular weight of about 27 kDa. GFP is derived from the crystal jellyfish Aequorea victoria. GFP can transform the blue fluorescence emitted by the jellyfish luminescent protein through chemical interaction into green fluorescence through energy transfer. GFP has an excitation wavelength of 488 nm and an emission peak of about 507 nm. At the same time, high-resolution crystal structure studies of GFP allowed scientists to manipulate its protein structure to create color variants that emit at different wavelengths [3,4]. The GFP antibody, protein, and ELISA kit family has been validated in a variety of critical applications, including Western Blot, immunohistochemistry, immunocytochemistry, chromatin immunoprecipitation (ChIP), flow cytometry, ELISA, and immune precipitation [2].

Figure 3. Gene Map of GFP

GAL4/UAS is a gene expression regulatory system present in yeast. UAS is the abbreviation of upstream activating sequence. GAL4 is a transcriptional regulator whose Binding domain (BD) binds to the UAS sequence, and its Activity domain (AD) binds to the promoter region, thus inducing gene expression. The GAL4/UAS system has been widely used in various gene regulation studies [4].

Figure 4. Gene Map of 2xUAS Promotor

Experimental Approach

First, the target gene fragments were extracted, and the pieces of GFP, UAS were amplified by PCR technology. After gel recovery, the target gene fragments were obtained by electrophoresis in agarose gel [5].

Figure 5. Target gene fragments of GFP, UAS

To add the target gene fragments into the BD scaffolds, it is necessary to use enzymes to cut out the gaps in the scaffolds. BD uses NdeI for single enzyme digestion [6].

Figure 6. The enzyme digestion of plasmid. Show the BD uses Nde1 for single enzyme digestion.

Through homologous recombination, GFP and UAS were added to the original BD skeleton and added to Escherichia coli (DH5α). To demonstrate the successful transfer of the skeleton and target fragment to DH5α, we added K+ resistance to BD and screened this by adding antibiotics K+ to the culture medium. When DH5α is grown, we also confirm the complete fusion of the skeleton with the target gene by colony PCR. After colony PCR results were obtained, it was proved that the bands after her PCR completion were consistent with the desired target bands, so we believed that the recombinant plasmid was constructed and could be correctly cloned and amplified within DH5α. After verification, the plasmid was extracted from DH5α for preservation [7].

Figure 7. Completed reconstruction of the plasmid. A shows the PCR amplification of BD-CRY2-N489-GFP. B shows the plasmid of BD-CRY2-N489-GFP.

To ensure that the plasmid construction is 100% correct, we sequenced the target genes that GFP [8].

Figure 8. Genetic sequence of GFP.

Characterization/Measurement

We used two methods to verify our results. One is a yeast two-hybrid, and the other is the β-galactosidase activity test.

Recombinant plasmids, done in previous steps, are connected by yeast mating. We build a hybrid system of experimental and control groups separately. For the experiment group, plasmid BD-CRY2-N489-GFP(UAS) is associated with AD-SPA1-N545 and transformed into yeast cells. For the control group, plasmid BD-CRY2 is associated with AD-CIB1 and transformed into yeast cells. Each group is tested by culturing them separately in -Trp-Leu and -Trp-Leu-His-Ade, and each culture dish is placed separately under dark and blue light conditions. Sensitivity is also tested by building a concentration gradient. At the same time, the sensitivity of the new blue light switch and the old blue light switch is compared [5].

Figure 9. Prove whether BD-CRY2-N489-GFP and AD-SPA1-N545 have successfully fused. A and C show the target plasmids BD-CRY2-N489-GFP and AD-SPA1-N545 successfully enter the yeast. B and D show the yeast that has turned the blue light switch needs blue light to grow, and the plasmids BD-CRY2-N489-GFP and AD-SPA1-N545 have successfully fused.

One of the differences between us and the old switch is that we added the GFP visualization gene to the new switch. When the blue light switch is successfully constructed, it will emit a bright green light when the blue light is illuminated. We used the BD+AD skeleton for no-load control, and the experimental group was a new blue light switch. We used blue light irradiation and white light irradiation to find that under white light irradiation, both groups could see white colonies but could not see the green fluorescence of GFP. When exposed to blue light, the experimental group could see green fluorescence of GFP, while the control group had a dark field of vision and no fluorescence. The experimental group fluoresces as evidence that the proteins can interact to make the GFP gene appear [10].

Figure 10. Test of GFP. Observe whether there is green fluorescence.

After proving successful plasmid fusion, sensitivity comparisons were made. The bacterial solution was diluted by ten times gradient and grew in -Trp-Leu culture medium under blue light. The results showed that with the decrease in concentration, yeast grew from vigorous to not growing under blue light. Later, at a concentration solution of 10^-4 to 10^-12, the quantity of yeast declined dramatically while the yeast with the new blue light switch continued to grow at 10^-12, and the yeast with the old blue light switch did not. This shows that the sensitivity of the new blue light switch is higher than that of the old blue light switch [11].

Figure 11. Sensitivity contrast. Shows the sensitivity of the new blue light switch is higher than that of the old blue light switch.

After we've done all the tests, we will test for beta-galactosidase activity. In the constructed plasmid, there is a gene Gal4 that can express β-galactosidase, and if the activity is high, this gene is successfully expressed. The colorimetric method was used in the test. Since β-galactosidase can produce p-nitrophenol, and p-nitrophenol has a maximum absorption peak at 400nm, the activity of β-galactosidase was calculated by measuring the increased rate of absorption value.

A standard curve is prepared before the beta-galactosidase activity test. The standard curve is established according to the absorbance of the standard tube (x, minus the OD value of the standard line with a concentration of 0) and the concentration (y, nmol/ml), and △A is put into the standard curve to calculate the amount of product generated by the sample (nmol/ml) [12].

Figure 12. Standard curve. The horizontal axis is concentration, and the vertical axis is absorbance.

Under blue light, the gene began to express, and beta-galactosidase activity increased over time. In contrast, genes were not expressed in the dark condition, and β-galactosidase activity did not change. Thus, it can be proved that the plasmid is successfully constructed, and the blue light switch is activated only in the blue light state, resulting in gene expression [13].

Figure 13. β-Galactosidase activity test. The change of β-galactosidase activity with time under blue light and dark.

References

1. Tien-Hung Lan, Lian He, Yun Huang, et al. Optogenetics for transcriptional programming and genetic engineering. Trends in Genetics, December 2022, Vol. 38, No. 12.
2. Jathish Ponnua, Tabea Riedela, Eva Pennera, Andrea Schradera, and Ute Hoeckera1. Cryptochrome 2 competes with COP1 substrates to repress COP1 ubiquitin ligase activity during Arabidopsis photomorphogenesis. A Botanical Institute and Cluster of Excellence on Plant Sciences, Biocenter, University of Cologne, 50674 Cologne, Germany.
3. Xu W, Bochen J, Lianfeng G, et al. A photoregulatory mechanism of the circadian clock in Arabidopsis. Nature Plants, 2021, 7(10).
4. Ge W D. Preliminary establishment of a technique for analyzing human gene function in the Drosophila GAL4-UAS system. Fudan University, Shanghai. 2000. DOI:10.7666/d.y362704.
5. Liu H, Gomez G, Lin S, Lin S, Lin C (2012) Optogenetic Control of Transcription in Zebrafish. PLoS ONE 7(11): e50738. doi:10.1371/journal.pone.0050738.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 2700
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2718
  Illegal BamHI site found at 888
  Illegal XhoI site found at 651
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1112
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 570
  Illegal BsaI.rc site found at 4255
  Illegal BsaI.rc site found at 7170
  Illegal SapI site found at 3172