Part:BBa_K1789015

GFP_N1

This device is a negative control of GFP with scaf.

Usage and Biology

This is the nagative GFP. Using the DNA-binding characteristic of the TAL effector, we can generate a new method to increase the production of

heterogenous multi-enzymatic reactions in Prokaryotic cells by rationally designed TALE proteins fused with specific

enzymes and their corresponding DNA sequences (as known as Binding Motifs [BMs]). Here we used Escherichia coli as our

chassis. A plasmid backbone was used as the scaffold for those BMs. The same plasmid was used to encode the fusion protein.

Thus, enzymes fused with TALE proteins could be gathered around the DNA scaffolds, enrich the local enzyme concentration,

and promote the rate of reaction.

This is the first experimental group of our project. Our theory is feasible if the functional parameter of this group is

stronger than the group of negative control.

Split GFP is a technique that has been widely used in the research of protein-protein interaction. In our project, we

demonstrated a prototype by fusing the Amino (or Carboxyl) Half of GFP with TALE1 (or TALE2/3).

By integrating the coding sequences of the TALE-fused proteins and the scaffold, three different plasmids can be

constructed and this is the second one.

Sequence and Features

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2311
  Illegal BamHI site found at 4908
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1304
  Illegal BsaI.rc site found at 3390
  Illegal BsaI.rc site found at 3798
  Illegal BsaI.rc site found at 4104
  Illegal BsaI.rc site found at 5094
  

Experimental Validation

This part is validated through four ways: Enzyme cutting, PCR, and Sequence

PCR

Methods

The PCR is performed with Premix EX Taq by Takara.

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

Results

Fig. 1 The result of the agarose electrophoresis was shown on the picture above.

Enzyme cutting

Methods

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 preparation of the plasmid was performed with TIANprep Mini Plasmid Kit from TIANGEN. The cutting procedure was performed with EcoRI and XbaI restriction endonuclease bought from TAKARA.

The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.

Results

Fig. 2The result of the agarose electrophoresis was shown on the picture above.

Functional Parameters

Functional check: Methodology: In order to investigate whether the fluorescence observed was due to the presence of the original GFP-N2 construction. First of all, the recombinant colonies were transferred into LB culture medium with chloramphenicol, and shook overnight at 37 ℃, then took the medium for PCR templet. Meanwhile, we extracted plasmid DNA using a miniprep kit and did a digest with Xbal1 and Pst1 to check for the presence of the original insert and size of the unfolded vector, respectively. Result and discussion: The DNA positive strand and the insert is very clearly visible at just below 3kb and 5kb. This proves the presence of GFP-N2 in culture.