Team:Chiba/Parts

Parts

    

Ferritin

Summary

    The ratio of FTH/FTL can be flexible in ferritin complex, and there exist a best composition that gives the highest Fe-storage activity. In heterologous expression of ferritin, the translational efficiency can be fine-tuned so that we could achieve that best composition. So, we constructed BioBricks for the functional expression of human ferritin complex in two formats;
BBa_K1057002: 'middle' RBS assigned for FTH
BBa_K1057009: 'strong' RBS assigned for FTH
In both construct, two ferritin genes (FTH and FTL) are placed under pBAD promoter to control the timing and expression level of these genes. To facilitate this construction process, we modified an existing BioBrick (BBa_I746908 deposited by iGEM 2007 team Cambridge) into the new BioBrick(BBa_K1057012). This enabled us the rapid, in-parallel, and one-pot digestion/ ligation using Golden gate method.

Fig. 1 Cloning procedure of ferritin-producing BioBrick

CRISPRi

Summary

    Recently, Qi and colleagues could show that a nuclease inactive mutant of Cas9 (dCas9) in combination with a sequence specific sgRNA can be utilized for targeted DNA recognition to interfere with transcriptional elongation, RNA polymerase or transcription factor binding (Fig. 2). With unique sgRNA specific for target region, you can knockdown target gene conditionally without any genome modification. This gene silencing activity was termed CRISPRi for CRISPR(clustered regularly interspaced short palindromic repeats) interference in reference to RNAi.

Fig. 2 CRISPRi mechanism

Improvement Parts : expression vector with pBAD/Ara switch compatible for "Golden gate gene swapping"

1.Introduction

     Gentic switch such as pBAD/araC system is very useful for overexpression of given genes. In order to place the various open reading frames with its RBS under the pBAD/araC system, we improved BBa_I746908 to insert BsaI site in both sides of sfgfp gene. This improvement enables us to use ‘Golden Gate’ cloning Method as described below (Fig. 3):
1) Preparation of insert fragment : Given gene(s) are PCR amplified with the additional sequence coding for ribosome-binding sites and BsaI site.
2) BBa_I746908 and PCR amplified insert fragment is BsaI digested and ligated in a single-pot reaction.
    This method is designed BsaI site doesn't remain on the vector after digesting BsaI. So, you can perform digestion and ligation at the same time. You can obtain desired plasmids in a short time.

Fig. 3 Efficiency of cloning for gene swapping (sfgfp is replaced by mrfp) at different insert/vector molar ration.

2.Material & Method

    We performed Golden Gate cloning with this part (vector) and mRFP (insert) and checked 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 cloning
3) transformation
Mixture list in Golden Gate is below.

Fig. 4 Mixture list

3.Results

1) The highest cloning efficiency (68.9 %) was obtained when the insert/vector molar ratio was 1:1. We suppose that the digestion (BsaI) efficiency could decrease with excess amount of insert (resulting in more "non-digested" green fluorescent colonies).
2) We found no non-fluorescent colonies in all tested conditions.
3) When we want to swap sfgfp to another gene with no phenotypic change, we could screen the right clone as non-fluorescent clones.

(A)
(B)

Fig. 5 Efficiency of cloning for gene swapping(sfgfpto mrfp), (A) percentage of recombinant and non-reconbinant clones (N.D. Not Detected) (B) colony fluorescence