Difference between revisions of "Part:BBa K1124005"

Latest revision as of 13:40, 5 October 2013

MicC sRNA scaffold (w/o antisense)

This is a template for BioBricked sRNA synthesis. You can make your own artificial sRNA BioBrick part to knock down a target gene.

How to make sRNA BioBrick part

1, Choose a target gene you want to repress.
2, The design principle described on our wiki(currently under construction) gives you PCR primers design. 
3, PCR (template: this BioBrick part (pSB1C3 carrying BBa_K1124005), primers: primers you get in 2.)
4, You get your sRNA BioBrick part. (the sRNA BioBrick parts UT-Tokyo 2013 made; BBa_K1124006, BBa_K1124007 ,BBa_K1124008, BBa_K1124009, BBa_K1124010 )

You can use this sRNA BioBrick part as follows;

1, Add a promoter and terminator.
2, You get sRNA generator of BioBrick standard.
3, Transform E. coli with sRNA generator BioBrick part. You will see knock down of the target gene.

sRNA (small RNA)

sRNA repress the translation by blocking TIR(*1) and by facilitating degradation of mRNA by RNase E [1]. Artificial sRNAs can be used in many ways (e.g. metabolic engineering, construction of genetic circuit,......).

(*1): TIR = translation initiation region

MicC

MicC is a sRNA of E. coli, which scaffold is used as a template for artificial sRNA synthsis [2].

Improvements

This is the improved part of BBa_K252001 from Illinois team in 2009 (https://parts.igem.org/Part:BBa_K252001 ) and BBa_K864440 from Uppsala team in 2012 (https://parts.igem.org/Part:BBa_K864440 ).

The problem in previous part (BBa_K252001): no experimental characterization, limited use (only one target gene, ompC)

The problem in previous part (BBa_K864440): cost much time, money, and labour because it requires screening.

Improvements: We have characterized the part experimentally (https://parts.igem.org/Part:BBa_K1124005 ). The part has broader versatility (You can choose a target gene to knock down). The part is easier to use (You can create BioBrick sRNA part by single reaction of PCR. The primers for PCR can be designed automatically with the tool shown on our wiki page (http://2013.igem.org/Team:UT-Tokyo/Project#RNA_Silencing )).

Characterization

Fig.1 Quantitative PCR analysis for tyrR mRNA

Fig. 2 growth curve

Fig.1 shows that anti-tyrR causes the decreasing of the relative concentration of the tyrR mRNA. 16s rRNA is used for internal control. We proved that the MicC scaffold and our sRNA design with pConst-sRNA(anti-tyrR) functions by qRT-PCR.

Fig.2 shows the growth curves. The vertical axis is for the OD600, and the horizontal is for time[hour]. The red curve is the growth curve of anti-tyrR(+), and the blue is one of anti-tyrR(-). This graph says that the anti-tyrR small RNA repress the E. coli's growth, but we cannot tell it is caused by the repression of tyrR or by off-target repression.

References

[1] Aiba, H. (2007). Mechanism of RNA silencing by Hfq-binding small RNAs. Current opinion in microbiology, 10(2), 134-139.

[2] Na, D., Yoo, S. M., Chung, H., Park, H., Park, J. H., & Lee, S. Y. (2013). Metabolic engineering of Escherichia coli using

synthetic small regulatory RNAs. Nature biotechnolog

Sequence and Features