Team:Chiba/Parts

Parts

DNA Biobrick

1. Introduction

    If you use pBAD promoter, you can regulate the expression and over expression. In order to combine the various parts, we improved BBa_I74608. We inserted BsaI site in both sides. Owing to this improvement, we were able to use Golden Gate. This part is designed BsaI site doesn't remain on the vector after digesting BsaI. So, if you design insert so as not to remain BsaI site, you can perform digestion and ligation at the same time. You can obtain desired plasmids in a short time.

2. Material & Method

    We performed Golden Gate with this part (vector) and mRFP (insert) and checked the function. And we investigated the reaction rate changing mol ratio of vector to insert. The protocol is below.
1) PCR up insert with BsaI site
2) Golden Gate
3) transformation
Mixture list in Golden Gate is below.

3. Result

    In the traditional ligation, the best ratio is vecor: insert= 1: 3. However, according to this experiment, the best ratio was vector: insert= 1: 1in the Golden Gate. The vector ............................... vectorが切られたあと再びsfGFPとligateする可能性もあり，これがまた切られるためにBsaIが使用される。したがって，insertが多いとvectorとBsaIの衝突頻度が低下するため，liationが進みにくいと考えられる。

    The maximum reaction rate was 68.9%. There existed few back ligations. Therefore, selecting colonies not shining green, you can pick the desired colonies easily.

CRISPRi

1.Introduction