1.
Overview
Light dependent optogenetic switch regulates gene expression, which is an important tool in synthetic biology, targeted
therapy of diseases and other fields (Lan et al.; Liu et al.). Among optogenetics tools, the blue light dependent CRY2/CIB1 switch has a wide
range of applications.
In the blue light dependent optogenetic switch, CRY2/CIB1 switch has been
developed for many applications. But because it can’t self-regulate the process of “off”, the
sensitivity of this system is not high enough compared with red light optogenetic switch. It needs to be
further optimized. So we focus on the newly discovered ultraviolet light receptor UVR8 in plants, which
transforms from an inactive dimeric form to an activated monomer form after UVB irradiation
(Rizzini et al.).
Table 1 Part co
llection
|
Parts number
|
Parts name
|
Contribution type
|
|
BBa_K4874000
|
TRP1 promoter
|
new
|
|
BBa_K4874001
|
UVR8-N397
|
new
|
|
BBa_K487400
2
|
new
|
|
|
BBa_K4874003
|
pbridge-BD
|
new
|
|
BBa_K4874004
|
pbridge-BD-CRY2(UVR8N-BIC2)
|
Add new info
|
2.
Create a New Part
2-1
BBa_K4874002, BIC2-CDS
BIC2 is a newly discovered CRY2 photoactivation inhibitor. It
can regulate the blue-light dependent dimerization of CRY2 and formation of photobodies and inhibit CRY
phosphorylation. BICs play two roles in CRY2 inhibition. On
the one hand, BIC2 inhibits the photoreduction of CRY2. The binding of BIC2 does not alter the
overall structure of the CRY2 PHR or FAD binding, but increases the distance between FAD and
residues in the electron transfer pathway and rotates the side chain of the proton donor, D393, by
approximately 52°
, yielding a longer d (HD393-N5ISO). Given the important role of D393 in
the photoresponse, the researchers suggest that this alteration may prevent the protonation of FAD
by D393, thereby interfering with the blue-light-dependent photoresponse of CRY2.
2-2
BBa_K4874001, UVR8-N397
UVR8-N397 DNA sequence could express UVR8 protein that acts
as UV-B photoreceptor and plays a key role in establishing UV-protective responses in plants. Upon UV-B
irradiation, UVR8 undergoes an immediate switch from homodimer to monomer, accumulates in the nucleus
and regulates the expression of the transcription factor by associating with chromatin. UVR8 is involved
in controlling aspects of leaf growth and morphogenesis in response to UV-B, is required for normal
progression of endocycle and has a regulatory role in stomatal differentiation.
3.
Add New Information to an existing Part
a) Construction of the plasmid pBridge-BD-CRY2(UVR8-BIC2)
We designed a plasmid containing CRY2 and UVR8-BIC2
fusion protein. To construct the plasmid pBridge-BD-CRY2(UVR8-BIC2), we selected
TRP as the promoter, PGK1 as the terminator of UVR8-BIC2; ADH1 as the promoter, and ADH1 as the
terminator of CRY2 within the plasmids. The TRP, PGK1 and UVR8N397 DNA fragments were amplified by PCR.
Then we performed homologous recombination to link the 3 DNA
fragments to the linear pBridge-BD-CRY2 vector digested by enzyme.
A schematic diagram of the plasmid construction can be
shown as follow (Fig.1).
Figure 1 pbridge-BD-CRY2(UVR8N-BIC2) plasmid construction
We transformed the plasmid into E.coli DH5a and cultured overnight on the solid
LB culture medium. Then we confirmed that the plasmid contained TRP-UVR8-BIC2 by bacteria PCR. The
length after colony PCR should be the sum of TRP UVR8 and BIC2, which equals 1731 bp. On plate 1,
electrophoretic strips 2,3, and 4 are successful and on plate 2, electrophoretic strips 1,3, and 5 are
successful. (Fig.2)
Figure 2. Colony PCR results.
Figure 3. The sequence data of the recombinant plasmid
pBridge-BD-CRY2(UVR8-BIC2).
To verify whether the plasmid was constructed
successfully, we picked our single colony and sent them to company for DNA sequencing, the final results
indicated that there were not genetic mutations on our genes and the recombinant plasmid in lane 4 of
plate 1 was successfully constructed validated by sanger sequencing (Fig.3).
b) Functional test
The homologous recombination plasmids of pBridge-AD-CIB1, pBridge-BD-CRY2 and
UVR8-BIC2 were transferred into AH109. We used SD double deficient medium, and yeast without
inserted DNA fragments lacked Leucine and Tryptophan, making it unable to grow. We designed the detection of β galactosidase activity. Essentially, the quick
“off” characteristic of UVR8 can be verified if the enzymatic activity shows a rapid decrease trend,
vice versa. Therefore, we set four different light conditions: dark, dark with UV, blue light, and blue
light with UV, and observed different performances of yeast cells with plasmids transformed.
Figure 4. Bar chart and line chart of β-galactosidase (β-GAL) activity
assay
As shown in figure 4, the activity of β-Gal was low
under dark and dark with UV. So we believed that pBridge-BD-CRY2(UVR8-BIC2) system did not work under
these consitions. Under blue light condition, the activity of β-Gal was increased with time. While under
blue with UV light condition, the increasing rate of β-GAL activity in the presence of UV was slower
than that in the absence of UV, indicating that the proteins UVB and BIC2 play the role of self-switch
off and improve the switch “off” in pBridge-BD-CRY2(UVR8-BIC2) system.
Reference
Lan, T. H., et al. "Optogenetics for Transcriptional
Programming and Genetic Engineering." Trends Genet 38.12 (2022): 1253-70. Print.
Liu, H., et al. "Optogenetic Control of Transcription
in Zebrafish." PLoS One 7.11 (2012): e50738. Print.
Rizzini, L., et al. "Perception of Uv-B by the
Arabidopsis Uvr8 Protein." Science 332.6025 (2011): 103-6. Print.