"
Team:Chiba/Parts
From 2013.igem.org
Parts
Golden Gate
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.
Figure 1. Golden Gate mechanism
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
Table 1. cfu/transformation
Table 2. Reaction rate
Figure 2. Reaction rate
Figure 3. Plate
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
One of the immune system is CRISPR (clustered regularly interspaced short palindromic repeats). Cas9 protein and sgRNA (small guide RNA) combine specific sequence and cut it. Using a modified Cas9 lacking endonucleolytic activity, we can use CRISPR as repressor. This system is CRISPRi (CRISPR interference) as shown in Figure 1. Designing guide region of sgRNA and coexpress dCas9, you can knockdown target gene conditionally.
