Overview

It is known that the length of anti-sense is related to its repression efficiency (N Nakashima et al., 2006^[1]), but the detail of relations between the length of anti-sense and their repression efficiency is still unclear. In this study, we made different lengths of anti-sense sequence (Fig. 1), and measured it's repression efficiencies.

Fig. 1 Each anti-sense repress mRNA.

Our experiments of anti-sense RNA (asRNA) sequence and it's length reveals the relation. Thus our findings will be a clue for other iGEMers that wants to design their own anti-sense sequence.

Introduction

In repressing gene by anti-sense, it is important to choose anti-sense length. However, it is difficult to decide anti-sense length. Theoretically, even if they are too long, it doesn’t repress target RNA effectively. The reason is because RNA polymerase takes a lot of time to synthesize them, and the diffusion rate of them is also made to be low. However, too short asRNA also has some problem. The short anti-sense cannot bind to the specific part of mRNA because it has too short complementary sequences of target RNA. In the industrial and academic places, it is hoped to use anti-sense that has suitable repression efficiency. For example, you can create knock down recombinant organisms easily by using strong anti-sense, and in iGEM, you can make a bio-devices which has a complicated gene network and require fine-tuned gene expression. Gene’s expression is not only ON or OFF. As stated above, each cases need each repression efficiency. Researchers currently try to change anti-sense repression efficiency by changing anti-sense’s binding sequence. However, it is found that this method is difficult.

In this study, we made many kinds of anti-sense which have different lengths, and are investigated to have different repression efficiency. These anti-sense constructs themselves is not useful for you. However our information is very useful. In our way, you can create the needed anti-sense. We expect this results will help you decide anti-sense sequences. It is our satisfaction that our study tells scientists and iGEMers that repressing by anti-sense is easy and acurate.

How to synthesize anti-sense constructs

Insert fragments were synthesized based on BioBrick by PCR. Forward primers are common (XhoI-Ptet (-10)). The primer binds to -10 region of Ptet (BBa_R0040) , and its end has XhoI restriction enzyme site. Each reverse primers are different (as90 NcoI, as120 NcoI) (Fig. 1). These primers bind to each special part of mRFP (BBa_E1010) ,and their ends have NcoI restriction enzyme site. Then we got various length insert fragments, as90 and as120. As90 is the anti-sense that covers 90 bp of mRNA, and as 120 is the anti-sense that covers 120 bp of mRNA (complement RBS and a part of mRFP sequence.) Of course, the sides of insert fragment have restriction enzymes XhoI, NcoI sites.

Fig. 1 Synthesizing anti-sense by PCR

Fig. 2 Ligate the insert fragment with H-stem vector

After we finished synthesizing insert fragments, we cut them and H-stem vector (our anti-sense expression vector) by XhoI and NcoI. Finally, we ligated them. The anti-sense constructs are complete because the insert fragments are ligated reversely. Then, we measured their repression efficiencies. As the same way, we made as30, as60 on H-stem vector and anti-sense B0034 examination. We performed repression examination by using their 4 anti-sense constructs.

How to assay

We selected mRFP for the target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense constructs and target gene was used for assay.

1. To cultivate the colony in 4 mL LB culture for about 20 hours
2. To control turbidity up to 0.1 at OD600
3. To cultivate the colony in 2 mL M9ZB culture for 9 hours (100mM IPTG induces antisense constructs, addition 20 uL)
4. To measure fluorescence after 9 hour

Future Work

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 work, 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 (BBa_R0040), and its 3 ‘ contains XhoI recognition site that is imperative to ligate with our anti-sense vector (H-stem vector). 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 (BBa_E1010)’s 400 downstream.
     PCR products that are amplified by these 2 primes showed in Fig. 1.

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 H-stem vector. 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.

1. N Nakashima et al. (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli. Nucleic Acids Res 34: 20 e138