Coding

Part:BBa_K4904001

Designed by: Zhu Tianyu   Group: iGEM23_SubCat-Peking   (2023-07-27)


SPA1（N545）


SPA1(N545)

Basic Part: BBa_K4904001(SPA1-N545)

Profile

  • Name: SPA1(N545)
  • Base Pairs: 1635 bp
  • Origin: Plants, Synthetic
  • Properties: SPA1(N545) is one of the three major negative regulators of Arabidopsis photomorphogenesis.

Usage and Biology

SPA1 is one of the four SPA quartet genes (SPA1, SPA2, SPA3, and SPA4) that play partially redundant functions in Arabidopsis [1-2]. All SPA quartet proteins interact with CRY2, although SPA1 showed the most robust interaction with CRY2 in response to blue light. We focused on the analysis of CRY2-SPA1 interaction for the rest of this study. 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 [3-4]. PHR is the evolutionarily conserved chromophore-binding domain; CCE is an effector domain that interacts with COP1 [5–9]. 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 [10], whereas the coiled-coil domain and the WD-repeat domain interact with COP1 and the COP1 substrates, respectively [11-12]. 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 1
Figure 1. 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 2
Figure 2. 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 3
Figure 3. The enzyme digestion of plasmid. Show the 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 4
Figure 4. 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 5
Figure 5. 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-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. The results showed that yeast in the experimental and control groups grew successfully in the -Trp-Leu culture medium (Fig 8A and 8C) and -Trp-Leu-His-Ade culture medium under blue light conditions (Fig 8B). In the dark, the yeast grew successfully in the -Trp-Leu dish but not in the -Trp-Leu-His-Ade culture medium (Fig 8D). The results showed that the two plasmids were successfully combined.

Figure 6
Figure 6. 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.

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 7
Figure 7. Sensitivity contrast. Shows the sensitivity of the new blue light switch is higher than that of the old blue light switch.

References

  1. 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]
  2. 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]
  3. 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/
  4. 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]
  5. Lin C, Shalitin D. Cryptochrome structure and signal transduction. Annu Rev Plant Biol. 2003;54:469–496. [PubMed: 14503000]
  6. 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]
  7. 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]
  8. 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: 11509693]
  9. Yang HQ, Tang RH, Cashmore AR. The signaling mechanism of Arabidopsis CRY1 involves direct interaction with COP1. Plant Cell. 2001; 13:2573–2587. [PubMed: 11752373]
  10. Yang J, Wang H. The central coiled-coil domain and carboxyl-terminal WD-repeat domain of Arabidopsis SPA1 are responsible for mediating the repression of light signaling. Plant J. 2006; 47:564–576. [PubMed: 16813572]
  11. 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: 14597662]
  12. Hoecker U, Quail PH. The phytochrome A-specific signaling intermediate SPA1 interacts directly with COP1, a constitutive repressor of light signaling in Arabidopsis. J Biol Chem. 2001; 276:38173–38178. [PubMed: 11461903]


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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 951
    Illegal SapI.rc site found at 1312


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