Difference between revisions of "Part:BBa I715019:Experience"

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<partinfo>BBa_I715019 AddReview 4</partinfo>
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<I>NUDT_CHINA 2016</I>
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We further optimized the best expression condition for this part, and introduced a new split-GFP system based on the sequence provided by this part. See the part main page for more information.
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Latest revision as of 08:36, 21 October 2016


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_I715019

User Reviews

UNIQdbf6f1a22e90a201-partinfo-00000000-QINU



••••

NUDT_CHINA 2016

We further optimized the best expression condition for this part, and introduced a new split-GFP system based on the sequence provided by this part. See the part main page for more information.

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••••

NUDT_CHINA 2015

A pair of primers that can extent the usage of this part

In our project, we plan to fuse the N-terminal of GFP (A.K.A. GFP1) to the C-terminal of TALE1 protein (you may visit our wiki for more details). However, the sample of BBa_ I715019 provided by the 2015 DNA distribution does not have a termination codon on its 3’ terminal to stop the translation. To fix this, we designed a pair of primers to add a termination codon on the 3’ terminal of BBa_ I715019 to further extend its usage.

F-Prime: 5’- GAATTCGCGGCCGCTTCTAGAATGC-3’

R-Prime: 5’- GGACTAGTATTATTGTTTGTCTGCC-3’


Functional Test

We also used the improved version (using the primers above) of GFP1 and GFP2 (the C- terminal of GFP, that part is also improved) and tested the their functions. In our experiment, GFP1 was fused with TALE1, GFP2 was fused with TALE2; and SCAF2 was added into the plasmid. The plasmid was transformed into E.coli BL21(DE3) and cultured in LB with 30mg/ml Chloramphenicol to OD600=0.6, then inducted with 1mM IPTG overnight.

Function of split GFP.jpg

Fig. 1 Evaluation of the functions of split GFP. 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 S2 control group 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 existence of GFP1 and GFP2 can observably increase the fluorescent intensity. Which then indicates that these two parts work as expected.

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