Team:HokkaidoU Japan/Projects/asB0034/Method

From 2014.igem.org

Revision as of 11:58, 14 October 2014 by Kakurehana (Talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

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