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 "POK-ROK Primer Designer"!

Promoter Selector

What our kit contains

Our promoter optimization kit 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.

fig.1

The color expression is induced by IPTG. If you don't need the color expressing construct, you can remove it by using PstI. LacZα reporter is placed between two BsaI sites(fig.2). The LacZα expressing construct will be replaced by chosen sequence using BsaI.

fig.2

How it works

fig.3

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

2. Digest and ligate your protein coding sequence and all our POK 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

3. You should get the construct shown below.(fig.5)

fig.5

4. Transform the ligated DNA to E. coli, and spread it on plate containing IPTG. Then, you will get colonies with five colors. (fig.6) The colors are paired with the promoters, so you will know which promoter is used instantly!

fig.6

5. Pick up the colonies and add to culture. Assay to check the production of protein. When you don't want the colors to be expressed, you can remove the color expressing construct by chosen sequence using PstI.(fig.7)

fig.7

RBS Selector

What our kit contains

Our kit contains tandem RBS (fig.8) and acceptor plasmid (fig.9)

fig.8

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 are connected together.

fig.9 This is 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, our program should help your primers design (fig.10). Also when you want to optimize more than one protein coding sites, add BsaI sites and overhang to them too. Be careful not to choose the same overhangs.

fig.10

2. Digest and ligate your protein coding sequence and all ROK together. This reaction is also accomplished by Golden Gate Assembly. DNA fragments will be assembled in the desired order (fig.11).

fig.11

3. Transform the ligated DNA to E. coli. If you are optimizing 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