Team:HokkaidoU Japan/Shuffling Kit/How To Use

From 2013.igem.org

Maestro E. coli

Shuffling Kit

How to use

What users should prepare

To use the kit, the protein sequence you chose must have specific prefix/suffix which contains a BsaI site and produce overhang. Therefore, users must design a primer to add it. To reduce time and trouble, we automated the design by creating program "Primer Designer for Maestro"!

Promoter Selector

What our kit contains

Our Promoter Selector consists of 5 different plasmids (table.1). Each has a promoter with a different strength. Downstream of the RBS there is a BsaI site to insert the protein sequence. There is a color expression construct downstream of protein insertion site (fig.1). Each color is paired with different strength promoter. The pairings are shown on the table below.

Part numberPromoterPromoter strengthPaired proteinProtein color
BBa_K1084501BBa_K1084001StrongestamilGFPyellowish green
BBa_K1084502BBa_K1084002StrongeraeBluestrong blue
BBa_K1084503BBa_K1084005MediumamilCPPurple
BBa_K1084504BBa_K1084009WeakermRFPPink
BBa_K1084505BBa_K1084010WeakesteforREDred
table.1 Matching list of promoters and their colors.
fig.1 The matching color for respective promoters.

The color always expresses. LacZα reporter is placed between two BsaI sites (fig.2). The LacZα expressing construct will be replaced by chosen sequence using BsaI.

fig.2 Insertion of LacZ╬▒ reporter between two BsaI sites.

How it works

fig.3 Digestion by BsaI.

1. Have BsaI site and specific overhang added to your protein sequence (fig.3). PCR with primers designed with Primer Designer for Maestro should do the trick.

2. Digest and ligate your protein coding sequence and all our Promoter Selector together (fig.4). This is accomplished by "Golden Gate Assembly" reaction. The detailed recipe is shown in Engler (2009)[1]. All the protein coding sequence will be inserted in the plasmid.

fig.4 How to insert CDS into protein coding sequence.

3. You should get the construct shown below after Golden Gate Assembly (fig.5).

fig.5 Constructs you can get.

4. Transform the ligated DNA to E. coli, and spread it on plate. Then, you will get colonies with five colors easily because you can see 5 colors by naked eyes (fig.6). The colors are paired with the promoters, so you will know what promoter you are using without sequencing all the colonies. You can pick up colonies and have an assay.

fig.6 Method after transformation.

RBS Selector

What our kit contains

Our kit contains tandem RBS (fig.7) and acceptor plasmid (fig.8).

fig.7 The sequence of tandem RBS.

In this part, 4 strength levels of RBSs[BBa_K1084101, BBa_ K1084102, BBa_ K1084103, BBa_ K1084104] are connected in tandem. To optimize up to 3 coding sequence expressions in the operon this part has three sets of RBSs with different overhangs and they are connected together.

fig.8 The acceptor part of the RBS and protein coding region. It has a BsaI site for the parts to be assembled.

How to use

1. Have BsaI site and specific overhang added to your protein sequence. Again, Primer Designer for Maestro should help your primers design (fig.9). Also when you want to apply more than one protein coding sites, add BsaI sites and overhang to them too. Be careful not to choose the same overhangs.

fig.9 Insertion CDS anf digestion by BsaI.

2. Digest and ligate your protein coding sequences and all RBS Selector together. This reaction is also accomplished by Golden Gate Assembly. DNA fragments will be assembled in the desired order (fig.10).

fig.10 How to use Golden Gate Assembly.

3. Transform the ligated DNA to E. coli. If you are using three different proteins, you will get 64 different kinds of constructs.

We will submit these standard methods as RFC to BioBrick Foundation.

  1. C. Engler et al. Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes (2009) PLoS ONE