Part:BBa_K4904003

AD-SPA1-N545

Composite Part: BBa_K4904003 (AD-SPA1-N545)

Construction Design

SPA1-N545 (BBa_K4904001) gene fragments were connected with AD (BBa_K4904005) no-load skeleton and added into Escherichia coli. Then the recombinant plasmid was extracted [1-2].

Figure 1. Map of AD-SPA1-N545.

Engineering Principle

SPA1 is one of the four SPA quartet genes (SPA1, SPA2, SPA3, and SPA4) that play partially redundant functions in Arabidopsis [3-4]. All SPA quartet proteins interact with CRY2, although SPA1 showed the most robust interaction with CRY2 in response to blue light. SPA1 Is Required for the CRY2-Mediated Photoperiodic Regulation of Floral Initiation. CRY2 has two domains, the N-terminal photolyase homologous region (PHR) domain and the C-terminal cryptochrome C-terminal extension (CCE) domain [5-6]. PHR is the evolutionarily conserved chromophore-binding domain; CCE is an effector domain that interacts with COP1 [7–11]. SPA1 is composed of three domains: the N-terminal kinase-like domain, the central coiled-coil domain, and the C-terminal WD repeat domain. It has been demonstrated that the kinase-like domain is a regulatory domain for SPA1 [12], whereas the coiled-coil domain and the WD-repeat domain interact with COP1 and the COP1 substrates, respectively [13-14]. SPA1 is an important group of negative regulators in the regulation of light signaling pathways. Studies using different mutants of SPA1 have shown that SPA1 mutants have no phenotype under dark conditions, suggesting that its inhibitory effect is dependent on light conditions and enhances photomorphogenesis. spa3 and spa4 mutant seedlings exhibit enhanced photomorphogenesis under sustained far-red, red, and blue light conditions. In addition, the yeast hybridization assay demonstrated that SPA3, SPA4, and COP1 can interact with each other, suggesting that they may take up some of the regulatory functions of SPA1 in the regulation of light signals. In addition, the protein structure of SPA1 is very similar to that of COP1, containing three functional domains, encoding a 114 kDa nuclear protein, a protein kinase-like domain at the N-terminus, a Coiled-Coil region in the middle, and a WD40 domain at the C-terminus. It is suggested that the Coiled-Coil and WD40 regions may be involved in the regulation of the inhibition of light signaling. Encodes a member of the SPA (suppressor of phyA-105) protein family (SPA1-SPA4). SPA proteins contain an N-terminal serine/threonine kinase-like motif followed by a coiled-coil structure and a C-terminal WD-repeat domain. SPA1 is a PHYA signaling intermediate, putative regulator of the PHYA signaling pathway. Light responsive repressor of photomorphogenesis. Involved in regulating circadian rhythms and flowering time in plants. Under constant light, the abundance of SPA1 protein exhibited circadian regulation, whereas, under constant darkness, SPA1 protein levels remained unchanged. In addition, the spa1-3 mutation slightly shortened the circadian period of CCA1, TOC1/PRR1, and SPA1 transcript accumulation under constant light.

Figure 2. Gene Map of SPA1(N545)

Experimental Approach

The target gene fragments were extracted, and the fragments SPA1 were amplified by PCR technology. After gel recovery, the target gene fragments were obtained by electrophoresis in agarose gel.

Figure 3. PCR amplification of SPA1.

To add the target gene fragments into the AD skeleton, it is necessary to use enzymes to cut out the gaps in the scaffolds. AD uses NdeI and BamHI for double enzyme digestion.

Figure 4. The enzyme digestion of plasmid. AD uses Nde1 and BamH1 for double enzyme digestion.

Through homologous recombination, SPA1 was added to the AD skeleton and added to Escherichia coli (DH5α). To demonstrate the successful transfer of the skeleton and target fragment to DH5α, we added Amp+ resistance to AD and screened them by adding antibiotics Amp+ 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 also constructed and could be correctly cloned and amplified within DH5α. After verification, the plasmid was extracted from DH5α for preservation.

Figure 5. Completed reconstruction of the plasmid. A shows the PCR amplification of AD-SPA1-N545 B shows the plasmid of AD-SPA1-N545

To ensure the plasmid construction is 100% correct, we sequenced the target genes that SPA1.

Figure 6. The genetic sequence of SPA1.

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 [15].

To connect and transform the plasmid into a yeast cell, we first preconditioned the carrier DNA by repeating heating and cooling it. The operation is done on a clean bench, ensuring the whole process undergoes a sterilized condition. Preconditioned carrier DNA, PEG/LiAC, and AH109 competence cells are mixed to construct the reaction system. BD-CRY2-N489-GFP and AD-SPA1-N545 are added to the experimental group, and BD-CRY2 and AD-CIB1 are added to the control group. Through centrifuge and resuspend, PEG/LiAC and inclusion are washed. Yeast containing those plasmids is then cultured in the -Trp-Leu culture dish if the successful growth in the -Trp-Leu culture dish indicated that BD-CRY2-N489-GFP and AD-SPA1-N545 were successfully transformed into DH5α.

In the next step, we need to prove whether BD-CRY2-N489-GFP and AD-SPA1-N545 have successfully fused, and only successful fusion can initiate downstream DNA transcription under the action of blue light and can successfully grow on the -Trp-Leu-His-Ade culture medium. Therefore, the experimental and control groups are cultured under low and blue light conditions. For each condition, each group was cultured in a -Trp-Leu dish and in a -Trp-Leu-His-Ade container under blue light and dark conditions, respectively.

Figure 7. 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.

Figure 8. 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.

Figure 9. 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).

Figure 10. 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.

Figure 11. β-Gal activity test. The change of β-galactosidase activity with time under blue light and dark.

Next steps

Chimeric antigen receptor (CAR)-T cell therapy is a revolutionary new pillar in cancer treatment. CAR-T has already shown a significant benefit in clinical service for patients with terminal cancer. This artificial T cell can identify cancer cells and use their hinge to identify the antigen of the cancer cell. After that, the T cell will release cytokine to activate the immune system to fight against cancer. However, this treatment faces various challenging problems. The over-release of cytokine can lead to a toxic level, thus causing several syndromes such as CRS, which refers to cytokine-release syndrome. CRS can be accompanied by cardiac dysfunction, system failure, and death. A recent method to ameliorate CAR-T toxicity is to use their gene strategies. However, this method is limited by its efficiency. Our new blue light-dependent interaction can add an “off-switch” on CAR-T cells. Since the interaction is operated by light, the interaction can give an immediate response and prevent the T cell from expressing more cytokine in time.

References

1. Tien-Hung Lan, Lian He, Yun Huang,2,* and Yubin Zhou. 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. Laubinger S, Fittinghoff K, Hoecker U. The SPA quartet: A family of WD-repeat proteins with a central role in suppression of photomorphogenesis in Arabidopsis. Plant Cell. 2004; 16:2293–2306.[PubMed: 15308756]
4. Zhu D, Maier A, Lee JH, Laubinger S, Saijo Y, Wang H, Qu LJ, Hoecker U, Deng XW. Biochemical characterization of Arabidopsis complexes containing CONSTITUTIVELY PHOTOMORPHOGENIC1 and SUPPRESSOR OF PHYA proteins in light control of plant development. Plant Cell. 2008; 20:2307–2323. [PubMed: 18812498]
5. Yu, X.; Liu, H.; Klejnot, J.; Lin, C. The Arabidopsis Book. Rockville, MD: American Society of Plant Biologists; 2010. The cryptochrome blue-light receptors. 10.1199/tab.0135, http://www.aspb.org/publications/arabidopsis/
6. Yu X, Shalitin D, Liu X, Maymon M, Klejnot J, Yang H, Lopez J, Zhao X, Bendehakkalu KT, Lin C. Derepression of the NC80 motif is critical for the photoactivation of Arabidopsis CRY2. Proc Natl Acad Sci USA. 2007; 104:7289–7294. [PubMed: 17438275]
7. Lin C, Shalitin D. Cryptochrome structure and signal transduction. Annu Rev Plant Biol. 2003;54:469–496. [PubMed: 14503000]
8. Brautigam CA, Smith BS, Ma Z, Palnitkar M, Tomchick DR, Machius M, Deisenhofer J. Structure of the photolyaselike domain of cryptochrome 1 from Arabidopsis thaliana. Proc Natl Acad Sci USA. 2004; 101:12142–12147. [PubMed: 15299148]
9. Yang HQ, Wu YJ, Tang RH, Liu D, Liu Y, Cashmore AR. The C termini of Arabidopsis cryptochromes mediate a constitutive light response. Cell. 2000;103:815–827. [PubMed: 11114331]
10. Kleiner O, Kircher S, Harter K, Batschauer A. Nuclear localization of the Arabidopsis blue light receptor CRY2 is associated with its function in light control of development. Plant J. 1999;19:641–650. [PubMed: 10504576]
11. Liu H, Yu X, Li K, Klejnot J, Yang H, Lisiero D, Lin C. Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis. Science. 2008;322:1535–1539. [PubMed: 19056982]
12. Wang H, Ma LG, Li JM, Zhao HY, Deng XW. Direct interaction of Arabidopsis cryptochromes with COP1 in light control development. Science. 2001;294:154–158. [PubMed: 11588270]
13. Saijo Y, Sullivan JA, Wang H, Yang J, Shen Y, Rubio V, Ma L, Hoecker U, Deng XW. The COP1-SPA1 interaction defines a critical step in phytochrome A-mediated regulation of HY5 activity. Genes Dev. 2003;17:2642–2647. [PubMed: 14522948]
14. Seo HS, Yang JY, Ishikawa M, Bolle C, Ballesteros ML, Chua NH. LAF1 ubiquitination by COP1 controls photomorphogenesis and is stimulated by SPA1. Nature. 2003;423:995–999. [PubMed: 12827201]
15. Ciro V, Maria MP, Paolo RC. Receptor and enzyme: the ambiguous identity of phytochrome B. Physiol Plant. 2019; 165: 33–41.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 4449
  Illegal NotI site found at 4470
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1897
  Illegal BamHI site found at 1971
  Illegal XhoI site found at 1983
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 210
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 860
  Illegal BsaI.rc site found at 5667
  Illegal BsaI.rc site found at 8892
  Illegal SapI site found at 4584
  Illegal SapI.rc site found at 9253