Part:BBa_K1789018
GFP_S1
This is a device which contains the sequence of the recombination of TALE1 and GFP1, the recombination of TALE3 and GFP2
and scaffold1. 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 second one.
Fig. 1 Split GFP fused with TALE1/TALE3 on SCAF1
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2311
Illegal BamHI site found at 4908 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 5323
Illegal AgeI site found at 5353
Illegal AgeI site found at 5383
Illegal AgeI site found at 5413
Illegal AgeI site found at 5443
Illegal AgeI site found at 5473
Illegal AgeI site found at 5503 - 1000INCOMPATIBLE 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_S1 plasmid except SCAF1. 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_S1. 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/TALE3-GFP2-Scaffold1 group was significantly higher than that of
no scaffold1 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-scaffold1 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.
References
None |