Future Works

In this results, we confirmed the repression efficiency of anti-sense is related to the length. Therfore, we can make many kinds of repression efficiency anti-senses by making some length anti-sense. However, to synthesize many kinds of anti-senses, we must prepare each primers. As a future works, we prpose an efficient method to synthesize various length anti-sense.

Method

Here, we explain this method by using mRFP expression construct as a target gene.

Preparation for randomizing

Fig.1 PrePCR for randomizing

Before randomizing, we have to perform some steps to make the effective anti-sense sequences . First, we performed PCR on mRFP construct to use below primers.

XhoI-pTet(-10)
mRFP 400dn

     XhoI-pTet(-10) is a primer that binds to -10 sequence of Ptet (R0040), and its 3 ‘ contains XhoI recognition site that is imperative to ligate with our anti-sense vector (H-Stem). Because it doesn’t contain -35 sequence, DNA synthesizing starts from Ptet’s -10 sequence and PCR products don’t contain a functional part as promoter.
     mRFP 400dn is a primer that binds to mRFP (E1010)’s 400 downstream.
     PCR products that are amplified by these 2 primes showed in figure ○.

Through this step, we can get about above 100bp fragments that seem the best length as effective anti-sense and contain SD sequence and start codon.

Randomizing

Next, we performed DNA synthesizing on PCR products that is synthesized some time ago as templates. The recipe showed below.

Fig. 2 Randomizing recipe

We added Klenow fragment, that is a DNA polymerase functioning on 37C, to the general PCR reaction system using KOD Plus NEO. We used 2 primers

XhoI-Ptet(-10)
NcoI-NNNNNN

NcoI-NNNNNN has random site contain any nucleotides (A, T, C, G), and they bind to random site of template. Its 3 ‘ contains NcoI recognition site that is imperative to ligate with our anti-sense vector (H-Stem). We explain these enzymes and primers’ function in any steps.
     In 37C step, Klenow fragment function. It synthesize DNA between XhoI-Ptet(-10) binding site and NcoI-NNNNNN binding sites that are random. DNA amplified in 3hrs are measurable length. This length is important for next step.

Fig. 3 How to make random anti-sense

Next, KOD Plus NEO starts general PCR system. In this reaction system, XhoI-Ptet(-10) and DNA fragment amplified by Klenow fragment work as primers. XhoI-Ptet(-10) bind to specific site of template DNA we hope, but another each primers bind to their specific random site because their sequences are different. Therefore we get some length PCR products.

Detail

We use Klenow fragment for reaction for NcoI-NNNNNN as primer. This primer is only about 6 mer that binds to DNA. Therefore in the reaction of KOD Plus NEO, it cannot anneal DNA because of high temperature. However, to use Klenow fragment and react slowly, DNA synthesizing that use NcoI-NNNNNN as primer becomes possible. By the way, Klenow fragment becomes deactivation through KOD Plus NEO’s reaction system.
     We used this DNA fragment as insert. We ligated them with the anti-sense vector, performed transformation in a tube and spread to a plate. Some inserts contain XhoI site and NcoI site at each ends and the other ones contain NcoI site at both ends. However, because after through dephosphorylation we ligated them, applied inserts were selected automatically in transformation.