How to synthesize anti-sense constructs

Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-senes RBS was flanked with scar sequences. Moreover, the ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. After finishing synthesizing anti-sense RNA, we ligated anti-sense RNA with H-stem construction by NcoI and XhoI.

Fig. 1 How to make anti-sense B0034 by primer annealing

Fig. 2 Using restriction enzyme, XhoI, NcoI, we made stem_anti-sense conplex.

Fig. 3 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site

Fig. 4 B0034 & B0032 sequence

Fig. 5 Our parts

How to assay

We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA 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 (IPTG induces antisense RNA, addition 20 uL)
4. To measure fluorescence after 9 hour

Fig. 6 Anti-sense B0034 is induced by IPTG