Part:BBa_K4016026

zdk-GFPnano

This composite part is designed to target GFP with Part:BBa_K4016025 through lov2-zdk interaction and GFP-GFPnano. With this composite part, zdk and GFPnano can be expressed in cells, and GFPnano was added to C-terminal of zdk.

Usage and Biology

In order to realize the assembly of the light control module, our team connected one basic part in light control module , zdk, with GFPnano, using the separation of aslov2-zdk under blue light and the combination in the dark, thereby indirectly through GFP fluorescence brightness to verify whether aslov2-zdk works.

LOV2 trap and release of protein (LOVTRAP), an optogenetic approach capable of repeatedly and reversibly controlling protein activity with precise kinetics. This approach uses a small protein, which we named Zdark (Zdk), generated by mRNA display screening of a library derived from the Z subunit of protein A. Zdk binds selectively to the dark state of LOV2[1]. Zdk is a kind of protein which can bind with lov2 induced by blue light.

GFPnano is the nanobody of GFP, a green fluorescent protein, which can be observed easily. This part could target GFP and bind TRIM21-aslov2 thought the aslov2-zdk interaction and lead to the degradation induced by ubiquitin-proteasome.

The PRYSPRY-lgG Fc interaction of (Part:BBa_K2653016) is replaced with aslov2-zdk interaction to realize controlling of both initiation and rate of the degradation. Zdk and aslov2 are proteins which separate from each other under induction of blue light. By providing dark environment, aslov2-zdk dimerization would trigger the formation of EGFP-GFPnano-truncated_Trim21 trimer, in which the truncated Trim21 would mediate the ubiquitylation and degradation of EGFP protein.

Figure 1. Schematic figure of BBa_K4016026 and BBa_K4016025

• Here is the mechanism of the recombined zdk-GFPnano:

1. zdk-GFPnano dissociate with HA-Trim21-aslov2 by the dissociation of zdk-lov2 under the light, and in the dark they connect together to work.

2. GFPnano binds with GFP, and target GFP.

Characterization

This part is validated through 4 experiments: PCR, enzyme digestion, sequencing and functional test.

PCR

The PCR is performed with 2 x Phanta Max Master Mix.

F-Prime: 5’-CTAGCGTTTAAACTTAAGCTTGCCACCATG-3’

R-Prime: 5’-TGGATATCTGCAGAATTCTTAtgaggagacggtgacctggg-3’

The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1％ Agarose glu.

Enzyme Digestion

After the assembly the plasmid was transferred into the Competent E. coli DH5α). After culturing overnight in LB,we minipreped the plasmid for cutting. The cutting procedure was performed with Hind III EcoR I restriction endonuclease bought. The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.

Sequecing

The plasmid was sequenced correct.

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 site found at 227
  

Functional test

This part (BBa_K4016026) was tested together with HA-Trim21-aslov2 (Part:BBa_K4016025).

Method

• 1. Florescent imaging:

To validate the function of our system(Trim21-aslov2-zdk-GFPnano), HEK-293T cells were co-transfected with GFP expressing reporter plasmids and our system expressing plasmids as the experimental group (plasmids: with the parts of Trim21-aslov2 and zdk-GFPnano). HEK-293T cells were co-transfected with GFP expressing reporter plasmids, the cells were divided into two groups. One was provided with blue light for 24 hours and the other was in the dark at the same time(control group). Florescent imaging showed that the different results of GFP fluorescence between our system and control group.

• 2. Dual luciferase reporter:

Moreover, we used a dual luciferase reporter (pEFR), in which firefly luciferase (Fluc) was fused with GFP, to obtain more accurate GFP abundance changes. Fluc activity was used as an indicator of GFP abundance, and the activity of a constitutively expressed Renilla luciferase (Rluc) reporter was used to normalize the noise caused by irrelevant factors such as transfection efficiency, cellular protein synthesis and cell growth.

(1） Cell Lysis Discard the cell culture medium and wash the cells twice with PBS. Add the appropriate amount of 1 × Cell Lysis Buffer as recommended (48-well: 50ul or 96-well: 20ul)Incubate or shake for 5 min at room temperature, pipette up and down and transfer the cell lysate to a 1.5 ml centrifuge tube.Centrifuge for 2 min, 12000 × g at room temperature.

(2) Firefly luciferase activity detection Add 100 μl of Luciferase Substrate (pre-equilibrated to room temperature) to the detection tube or microplate. Carefully pipette 20 μl of the cell lysate into the test tube or the plate. Mix rapidly and immediately detect the Firefly luciferase reporter gene activity using a luminometer or a full-spectrum microplate reader.

(3) Renilla luciferase activity detection Add 100 μl of freshly prepared Renilla Substrate solution to the above reaction solution. Mix rapidly and immediately detect the Renilla luciferase reporter gene activity.

Reference

[1] Hui Wang,Klaus M. Hahn. LOVTRAP: A Versatile Method to Control Protein Function with Light[J]. Current Protocols in Cell Biology,2016,73(1):