Overview

It is known that the length of anti-sense sequence is related to its repression efficiency (N. Nakashima et al., 2006^[1]), but the details of the relation is still unclear. In this study, we made different lengths of anti-sense sequence (Fig. 1).

Our experiments on anti-sense RNA (asRNA) sequence and its length reveals the relation. Thus our findings will be a clue for other iGEMers who wants to design their own anti-sense sequence.

Introduction

In repressing gene by anti-sense RNA, it is important to determine the length of anti-sense. However, it is difficult to do it. Theoretically, if the length is too long, it doesn’t repress target RNA effectively. The reason is because, the RNA polymerase takes a lot of time to synthesize them, and the diffusion rate of them also gets 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 fields, people hope 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 bio-devices which have a complicated gene network and require fine-tuned gene expression. Gene expression is not only ON or OFF. As stated above, each cases need each repression efficiency. Researchers currently tried to change anti-sense repression efficiency by changing anti-sense’s binding sequence. However, it is found that this method is difficult.

In this project, we made many kinds of anti-sense which have different lengths. These anti-sense constructs themselves is not useful for you. However, in our method, you can create the anti-sense that you desire. We expect this project will help you decide anti-sense sequences. We'd like to tell scientists and iGEMers how easy and accurate to repress the gene expression by anti-sense RNA.

How to synthesize anti-sense constructs

Insert fragments were synthesized based on BioBrick by PCR. As Forward primers, "XhoI-P_tet (-10)" was used for making all fragments. The primer binds to -10 region of P_tet (BBa_R0040) , and its end has XhoI restriction enzyme site. To change the down stream seqeunce, each reverse primers are designed differently (as90 NcoI, as120 NcoI) (Fig. 3). These primers bind to each specific part of mRFP (BBa_E1010) ,and their ends have NcoI restriction enzyme site. By that way, we can get various length of 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 edges of insert fragments have restriction enzymes XhoI, NcoI sites.

After we finished synthesizing insert fragments, we inserted them into our H-stem vector (anti-sense expression vector) by XhoI and NcoI. Then, we measured their repression efficiencies. In the same way, we made as30, as60 on H-stem vector and anti-sense B0034 experiment. We performed repression experiment by using their 4 anti-sense constructs.

How to assay

We performed RT-PCR to confirm the transcription of anti-sense RNA (asRNA) constructs.

1. Cultivate the colony in 4 mL LB medium for 16 hours.
2. Centrifuge the 4 mL of culture at 10,000 rpm / for 2 min / at 25°C
3. Remove the supernatant and add M9ZB medium then voltex the pelet.
4. Perform RT-PCR
5. Measure absorbance of 260nm about cDNA.

Results

Though we measured absorbance of 260nm about cDNA, we could not get any cDNA. After RNA extraction, we confirmed absorbance of 260nm (this is absorbance of nucleic acid). However, after RT-PCR of that prodocuts, we could not confirm the existence of nucleic acid.
Here, we show the discussion.

We estimated there is some problems in RNA extraction. First, we maybe loss RNA during the experiment operation. RNA is degraded easily than DNA because RNase is through the world, soil, air and water, of course in laboratories and human's bodies. We seemed to be careless for about it and overlook RNase's contamination.

Second, Deactivation of DNase seemed incomplete. We used DNase at the end of RNA extraction because extracted sample contain DNA and RNA. To do it, we removed DNA and get only RNA. After all steps of RNA extraction, we deactivate DNase at 65°C for 10 min. Probably, DNase was not deactivated. Because of it, DNase degraded cDNA produced in RT-PCR.

Though we could not be in wiki freeze, we are going to retry. We will perform it being careful in these problems.

Developmental experiment

We theoretically estimated 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 propose an efficient method to synthesize various length of anti-sense.

Method

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

Preparation for randomizing

Before randomizing, we have to perform some steps to make the effective anti-sense sequences . First, we performed PCR on mRFP construct. We call this step "prePCR". We used below primers.

• XhoI-P_tet (-10)
• mRFP 400dn

XhoI-P_tet (-10) is a primer that binds to -10 sequence of P_tet (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 P_tet’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. 5.

Through this step, we can get insert fragments containing SD (Shine-Dargalno) sequence and start codon that are important to effictive repression.

Randomizing

Next, for DNA synthesizing, we used PCR products synthesized previous step. The recipe showed below.

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

• XhoI-P_tet (-10)
• NcoI-NNNNNN

NcoI-NNNNNN has random site containing any nucleotides (A, T, C, G), and these random primers bind to random site of template. Theirs 3 ‘ contains NcoI recognition site that is imperative to ligate with H-stem vector. We explain function of these enzymes and primers in any steps.
In 37C step, Klenow fragment work. It synthesize DNA between XhoI-P_tet (-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.

Next, KOD Plus NEO starts general PCR system. In this reaction system, XhoI-P_tet (-10) and DNA fragment amplified by Klenow fragment work as primers. XhoI-P_tet (-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 contain NcoI site at both ends (Fig. 4). However, because after through dephosphorylation we ligated them, applied inserts were selected automatically in transformation.

Results

We performed an experiment by another recipe. Annealing temperture and PCR cycles differs. Low annealing temperture is better than high temperture because random primers have only 6 base binding region. it is important to synthesize various kinds of fragments that many random primers bind stably. To increase PCR cycles, fragments are amplified. The one side (Recipe A) was annealing temperture 55°C, PCR cycles 20cycles. The other side (Recipe B) was annealing temperture 40°C, PCR cycles 35cycles.
We got some colonies in both plates! More colonies existed in Recipe B than Recipe A. Then, we performed colony PCR about them. However, the result was not desired.

Discussion

We estimate 2 reasons that Recipe B had more colonies than Recipe A. First reason is, thanks to low annealing temperture, various random primers binded to target gene and amplified insert fragments. Second reason is in more PCR cycles, insert fragments were more amplified.
We performed this experiment in August and we treated the result was failure. Thus we gave up this experiment. However, we found that stem constructs are not PCRed well in the end of September. It is possible that the constructs were complete.

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