Part:BBa_K1789020

GFP_S2

This is a device which contains the sequence of the recombination of TALE1 and GFP1, the recombination of TALE3 and GFP2 and scaffold 1. Through this device, we can test and verify that our system is effective and correct.

Usage and Biology

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 first one.

Fig. 1 Split GFP fused with TALE1/TALE2 on SCAF3

This prototype is designed to test if our system can achieve our goal of compartmentation by examining if the green florescent intensity raised observably.

Fluoroskan Ascent FL by Thermo can be used to detect the fluorescence intensity.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 5323
  Illegal AgeI site found at 5385
  Illegal AgeI site found at 5447
  Illegal AgeI site found at 5509
  Illegal AgeI site found at 5571
  Illegal AgeI site found at 5633
  Illegal AgeI site found at 5695
  
• 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

Sequencing

This part is sequenced as correct after construction.

Split GFP Assay

To evaluate whether the plasmid DNA scaffold can bind the TALE protein efficiently and then increase the production of multi-enzymatic reactions in prokaryotic cells, we constructed a plasmid for negative control which is exactly the same with this GFP_S3 plasmid except SCAF3. These two plasmids were transferred into E.coli BL21(DE3) and cultured in LB with 30mg/ml Chloramphenicol to OD600=0.6, then inducted with 1mM IPTG overnight.

Fig. 2 Evaluation of the functions of GFP_S3. The green fluorescence (Ex: 488 nm; Em: 538 nm) of split GFP was detected after overnight culture of E.coli with or without GFP1/2 under the 1mM of IPTG induction. Relative fluorescence intensity was calculated with normalization of OD600 value. The relative fluorescence intensity of negative control group without IPTG induction was set arbitrarily at 1.0, and the levels of other groups were adjusted correspondingly. This experiment was run in three parallel reactions, and the data represent results obtained from at least three independent experiments. *0.01<p<0.05.

The results shows that the value of FI/OD600 in TALE1-GFP1/TALE2-GFP2-Scaffold3 group was significantly higher than that of no scaffold3 control.

RT-PCR Analysis

In order to prove that the increase of the green fluorescence in scaffold system was not owing to the expression variation of split GFP, we add a RT-PCR analysis.

Fig. 3 RT-PCR analysis for determination of GFP1 and GFP2 expression in TALE-GFP-scaffold3 groups. The cDNA sequence of 16S rRNA was amplified as standard.

These findings suggest that TALE-DNA scaffold system might be an efficient device for the compartmentation and ordering of different proteins fused with TALE proteins.