Part:BBa_K3734017

GIP-miR21T-GI-GAL4-4XmiR21T

GIP is a promoter that can be induced by glucose.And miR21T is miR21's target.When miR21 combined with miR21T, it will inhibit the expression of target gene.GI is a kind of light-activated protein. If you shine blue light on it, it will change its own structure and will be able to combined with LOV. GAL4 is a kind of protein that can find and combined with UAS DNA structure domain.

GIP-miR21T-GI-GAL4-4XmiR21T

GIP-miR21T-GI-GAL4-4XmiR21T consists of glucose-induced promoter GIP, photosensitive protein GI, double yeast hybrid system components GAL4 and miR21T.
It is regulated by both glucose concentration and blue
light, which improves the safety of the whole system. At the same time, it is also suppressed by miR21 feedback, which makes the whole system form a closed loop 
and makes the reaction more sensitive.

In order to increase the safety of patients' use, it may be difficult to achieve the expected goal of accurate regulation by relying on the regulation of blood glucose 
concentration alone, so we designed a pair of photosensitive proteins 
of GI ( BBa_K3734004) and LOV (BBa_K3734006). Under blue light irradiation, the Protein structure changes and interacts with each other to form a GAL4-GI-LOV-VP16 quadruple, while GAL4 ( BBa_K3734005) identifies and combines 9XUAS (BBa_K3734016), so that VP16 can activate the gene of downstream expression.

1.Pattern diagram

Fig.1 The model diagram of GIP-miR21T-GI-GAL4-4XmiR21T

2.1 Method

We used report gene LUC to represent the effect of GIP-miR21T-GI-GAL4-4XmiR21T

2.2 Result

Fig.2 Light controlled system testing experiment

Fig.3 Expression level analysis under blue light irradiation and dark treatment

3.Caution

The duration of GI and LOV after 450nm blue light exposure will vary with the irradiation method, intensity, etc. In the experiment, we adopted the method of irradiating strong light for 30 minutes first, and then low light for 30 minutes. The GAL4 binding domain is universal in eukaryotic cells, but transcription activators (such as VP16) that cooperate with GAL4 need to be properly selected according to different chassis organisms.

Reference：

[1]Chun Jeih Ryu , Charles E Whitehurst, Jianzhu Chen.Expression of Gal4-VP16 and Gal4-DNA binding domain under the control of the T lymphocyte-specific lck proximal promoter in transgenic mice[J].BMB Rep. 2008 Aug 31;41(8):575-80.

[2]Masayuki Yazawa , Amir M Sadaghiani, Brian Hsueh.Induction of protein-protein interactions in live cells using light[J].Nat Biotechnol. 2009 Oct;27(10):941-5.

[3]Kai Zhang , Xue-Jiao Yang , Ting-Ting Zhang.In situ imaging and interfering Dicer-mediated cleavage process via a versatile molecular beacon probe.Anal ChimActa.2019Nov4;1079:146-152.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 4624
  Illegal NotI site found at 1438
  Illegal NotI site found at 4997
  Illegal NotI site found at 5479
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 12
  Illegal BglII site found at 4519
  Illegal XhoI site found at 2172
  Illegal XhoI site found at 5229
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1127
  Illegal NgoMIV site found at 2674
  Illegal NgoMIV site found at 6055
  Illegal NgoMIV site found at 7397
  Illegal NgoMIV site found at 7680
  Illegal AgeI site found at 3901
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 5148
  Illegal BsaI.rc site found at 444
  Illegal BsaI.rc site found at 3298
  Illegal BsaI.rc site found at 9208
  Illegal SapI site found at 8125
  Illegal SapI.rc site found at 7246
  Illegal SapI.rc site found at 7456