Team:HokkaidoU Japan/Projects/asB0034
From 2014.igem.org
Overview
Anti-sense RNA (asRNA) is studied actively over the world. asRNA can be easily synthesized, but there is no clear method to make stable, highly efficient asRNA. It is a labor to design efficient asRNA for every target gene you want to repress.
As a solution, we decided to design a general asRNA which could repress various target genes.
To achieve this, we constructed an asRNA for RBS. For gene expression, RBS is indispensable and most of iGEMers use B0034 in their projects. Thus, we constructed and registered an asRNA for B0034 as a new part "Anti-sense RBS fragment B0034 (BBa_K1524107)". By this part, iGEMers can repress any target gene that is synthesised downstream B0034.
We also registered an anti-sense RBS fragment for B0032 "Anti-sense RBS fragment B0032 (BBa_K1524108)". You can repress the target gene individually by changing the combination of anti-sense RBS fragment and the target gene.
You can repress expression of your target gene without resynthesizing your constructs. All you have to do is to add our asRNA to the construct with the target gene!!
Method
The targets of these asRNA are B0034 (BBa_B0034) and B0032 (BBa_B0032). Both RBS are popular among iGEM. Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-sense RBS was flanked with scar sequences. The ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. Therefore, after the synthesis of anti-sense RNA, we can ligated asRNA with H-stem construct by NcoI and XhoI.
How to assay
We selected mRFP as the target gene. We used fluorophotometer to measure how the asRNA worked. The colonies transformed by asRNA and the target gene were used for the assay.
- Cultivated a colony of transformed bacteria in 2ml of LB medium (until the turbidity at OD600 reached 0.1).
- Retrieved the bacteria and cultivated them in 2ml of M9ZB medium
- Centrifuged the culture at 10,000 rpm / for 2 min / at 25°C
- Removed the supernatant and add M9ZB medium then voltex the pelet.
- Performed RT-PCR
- Measured absorbance of 260 nm about cDNA.
Results
We assessed the efficiency to suppress expression of two kinds of asRNA family: one has PT structure (Nakashima's stem) and pHN1257 as the vector, the other has newly designed H-stem structure. For each kind of asRNA, we prepared 4 types of E. coli:
- target B0034 + anti-sense B0034
- target B0034 + anti-sense B0032
- target B0032 + anti-sense B0034
- target B0032 + anti-sense B0032
We examined each sample with / without IPTG induction.
Nakashima's stem
We assessed whether asRNA with Nakashima's stem work. We used Nakashima’s plasmid (pHN1257[1][2]) as a vector. We double transformed separate plasmids of antisense and target gene and had an assay. All anti-sense constructs are on pHN1257 and all target constructs are on pSB6A1. The sample E. coli were cultivated for 18h in M9ZB medium.
Details of pHN1257 vector
This vector is published by Nakashima for transcribed anti-sense RNA. The feature of vector is Paired Termini (PT) structure. PT makes stem-loop construct and stabilizes anti-sense cassettes. The restriction enzyme (NcoI and XhoI) sites are between PT sites. The vector resistance is Kanamycin. Also, it has IPTG inducible promoter, Ptrc. Copy number is 30 (reprication origin is pSC101).
Fig. 11 shows the fluorescence strength ration between IPTG induction (+) / (-). smaller values indicate that the mRFP gene was suppressed because of asRNA, induced by IPTG. In the case of B0034, both asB0034 and asB0032 suppressed the target gene expression. However, for B0032, neither asB0034 nor asB0032 was confirmed to work. From these results, we were not able to confirm specificity of asB0034, but toward the construct using B0034, asB0034 down-regulated the expression by 40%, and asB0032 did by 80%. In Nakashima's results, the efficiency of down-regulation was 78%, so we got the same result.
H-stem
We inserted anti-sense between H-stem insted of PT structure and assayed their efficiency.
From these experiments, we were not able to confirm whether asRNA worked by IPTG induction using fluorescence intensity. So, we checked the expression of anti-sense using RT-PCR, and one with IPTG induction showed the expression of anti-sense. (However, we were unable to gain data of IPTG-.) From this result, we confirmed that, at least, asB0034 was expressed.
Discussion
We are assuming three possible causes of that H-stem system made no difference between the cases with and withou IPTG induction.- Anti-sense was not induced by IPTG; it is leaking. Seen from Fig. 12, fluorescence strength did not differ between IPTG+ and IPTG- for any sample. Since fluorescence showed no difference, it could be assumed that the asRNA was constantly over-expressed regardless of IPTG induction. Also, on Fig. 10, it was confirmed that asB0032 works on B0034 construct, but it was not functional on H-stem system. Therefore, because anti-sense was not under regulation of IPTG induction, we were not able to confirm the activity of anti-sense by fluorescence intensity.
- Antisense was not expressed sufficiently. From fig.10, we found little gap in fluorescence of mRFP for constructs using B0032 with/without IPTG induction. Likewise from fig.12, we found little gap either. In consideration of these facts, we guessed that asRNA expression were not sufficient. We confirmed the existence of antisense B0034 by sequencing the vector, though we did not confirm about antisense B0032. The reason is the difficulty to sequencing of DNA which has stem-loop stractures.
- Instability of copy number of target gene Seeing Fig.10 and 12, even if it were the same target genes, they sometimes had big differences in the degree of expression. By all rights, target gene under the control of B0034 which is stronger RBS should be larger degree of expression than that of B0032. But the result was completely opposite. Owning to making several assays, target gene increased little or in case, even if the same origin of plasmids, cultivate them from different colony, the expression of mRFP showed gap. Therefore, because of changes of copy number of target gene, expression of target genes weren’t enough and we found that it was difficult to measure and estimate the activation of antisense by fluorescence.
Improvement
- Analysis by RT-PCR By analyzing quantity of asRNA, we showed the expression can be induced using IPTG. We will anti-sense works even if copy number is unstable by comparing mRNA of target gene with / without anti-sense.
- Improvement of medium to induce anti-sense by adding IPTG easily To induce asRNA by IPTG easily, we have used M9ZB medium. However the medium includes glucose, IPTG induction may be too late. We will try to use LB medium to enable IPTG induction.
- Stability of copy number in target gene It was published that low copy plasmid often occur movement of copy number. We need to select plasmids with higher copy number. We will use medium included an 1.5 times antibiotic for screening.
We are going to show positive result in Boston!
Conclusion
We showed asRNA works in case using Nakashima’s stem. On the other hand, in case of H-stem system, we could confirm only transcription of antisense but we could not get a proof that antisense worked. We want to show some results in the presentation at Boston by re-thinking copy number of plasmids or medium for assay to work antisense even in H-stem.
Instability of mRFP expression
It’s possible to suppress 80% of registered parts in iGEM using RBS by asB0034 and asB0032. In short, it is very provable to be a common way of expression of proteins because it can suppress iGEM parts easily.
Reference
- N. Nakashima et al. (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli. Nucleic Acids Res 34: 20 e138
- N. Nakashima and T. Tamura (2009) Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli. Nucleic Acids Res 37: 15 e103