Overview

Using anti-sense RNA (asRNA) is one of the methods to repress gene expression. 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 are still unclear. In this project, we made different lengths of anti-sense sequence (Fig. 1).

Our experiments will be a clue for other iGEMers who want 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 problems. 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 case needs each repression efficiency. Researchers currently tried to change anti-sense repression efficiency by changing anti-sense’s binding sequence. However, it is known that this method is difficult.

In this project, we made many kinds of anti-sense which repress mRFP, but these anti-sense constructs themselves are 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.

Method

This is how to synthesize anti-sense constructs. We made anti-sense constructs that repress mRFP. The target construct are composed of P_tet (BBa_R0040), B0034 (BBa_B0034), mRFP (BBa_E1010) and double terminator (BBa_B0015 ) Insert fragments were synthesized based on BioBrick by PCR. As forward primer, ”XhoI-P_tet (-10)"”was used for making all fragments. The primer binds to -10 region of P_tet, and its end has XhoI restriction enzyme site. To change the downstream seqeunce, each reverse primer is designed differently (as90 NcoI, as120 NcoI) (Fig. 3). These primers bind to each specific part of mRFP, 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 BBa_K1524100) by XhoI and NcoI. Then, we measured their repression efficiencies. In the same way, we made as30, as60 on H-stem vector in other experiment (as30 (Anti-sense B0034) and as60 (H-stem system)). 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. Cultivated the colony in 4 mL LB medium for 16 hours.
2. Centrifuged the 4 mL of culture at 10,000 rpm / for 2 min / at 25°C
3. Removed the supernatant and add M9ZB medium then voltex the pelet.
4. Performed RT-PCR
5. Measured absorbance of 260 nm about cDNA.

Results

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

It is presumable that there are some problems in RNA extraction. First, we might have lost RNA during the experiment operation. RNA is degraded easily than DNA because generally RNase can be easily contaminated.

Secondly, the incomplete deactivation of DNase can be considered to be the reason of our failure. If DNase was not deactivated sufficiently, it will result in the degradation of cDNA produced in RT-PCR.

Though we could not make it by wiki freeze, we are going to retry this experiment.

Developmental experiment

We theoretically estimated that the repression efficiency of asRNA is related to its length. To find the optimum length, we must design many lengths of asRNA, and measure their efficiency. However, that takes a lot of time and labor. So, as a future work, we propose an efficient method to synthesize various length of anti-sense.

Method

Preparation for randomizing

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

• XhoI-P_tet (-10)
• mRFP 400 down

XhoI-P_tet (-10) is a primer that binds to -10 region of P_tet (BBa_R0040), and its 5’contains XhoI recognition site that is imperative to ligate with our anti-sense vector (H-stem vector). Because it doesn’t contain -35 region, DNA synthesizing starts from P_tet’s -10 region and PCR products don’t contain a functional part as promoter.
mRFP 400 down is a primer that binds to mRFP (BBa_E1010) 400 bp 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

For this method, we designed two primers shown below. The second primer is a "random primer" which can bind with any sequence on the template DNA. By using these primers, we can synthesize various lengths of anti-sense.

XhoI-P_tet (-10)
• NcoI-NNNNNN

However, the random primer only has 6 nt of region that anneals with the template DNA. Eventually, the Tm value of this primer gets extremely low. This causes some difficulties to PCR. As a solution, we have to take three unusual steps for PCR.
First, to single-strandize the template DNA, putting it at 95°C / for 3 min and then putting it in 0°C water. By this procedure, the template DNA will be kept denatured in low temperature.
Second, to anneal the primer and amplify the sequences in low temperature, we use "Klenow Fragment", a polymerase whose optimum temperature is 37°C. In this step, we can not amplify fragments, but can make templates of various lengths.
In the third step, we amplify the templates we mentioned above at 68°C just like the usual PCR.

By these methods, you can get various lengthes of asRNA of target gene. These fragments are all in one mixture of PCR products. Thus, by digesting, ligating the fragments into a vector and transforming them to cells, you can get various colonies with different lengths of anti-sense. The colony with the optimum length of anti-sense must be on the plate!

Next, for DNA synthesizing, we used PCR products synthesized previous step. The recipe is showed below. We denatured it to single strand by putting the template DNA at 95°C / for 3 min and put 0°C water.

We added Klenow fragment, that is a DNA polymerase functioning on 37°C. It synthesizes DNA as a single strand DNA. We had to denaure the template DNA for Klenow fragment because DNA is double strand at 37°C. Weput Klenow fragment to the general PCR reaction system using KOD Plus NEO. This is the polymerase of Themococcus kodakaraensis that is Hyperthermophiles. We call this Klenow working step "PCR adding Klenow". We used 2 primers.

XhoI-P_tet (-10)
• NcoI-NNNNNN

NcoI-NNNNNN has random site containing all nucleotides (A, T, C, G), and these random primers bind to random sites of a template. Theirs 5’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 37°C step, Klenow fragment work. It synthesizes DNA between XhoI-P_tet (-10) binding site and NcoI-NNNNNN binding sites that are random. DNA amplified in 3hrs by Klenow fragment are measurable length. These lengths are 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 desire, but another each primers bind to their specific random sites because their sequences are different. Therefore we can get some length PCR products. We ligated them and pHN1257 (anti-sense vector which is described "Anti-sense B0034").

Detail

We use Klenow fragment for reaction for NcoI-NNNNNN as primer. This primer is only about 6 nt 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 at 37°C, DNA synthesizing that use NcoI-NNNNNN as primer becomes possible. By the way, Klenow fragment becomes deactivation through KOD Plus NEO’s reaction system at 94°C.
We used these DNA fragments as insert. We ligated them with the H-stem vector, anti-sense vector, performed transformation in a tube and spread to a plate. Some inserts contain XhoI site and NcoI site at each edges and the other contain NcoI site at both edges (Fig. 8). However, H-stem has edges XhoI and NcoI, thus applied inserts were selected automatically in transformation.

Results

In fact, we performed an experiment by another recipe. Annealing temperature and PCR cycles differs. We think low annealing temperature is better than high temperature 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 more amplified. The one side (Recipe A) was annealing temperature 55°C, PCR cycles 20 cycles. The other side (Recipe B) was annealing temperature 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 estimated 2 reasons that Recipe B had more colonies than Recipe A. First reason is, thanks to low annealing temperature, 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.

Reference

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