Part:BBa_K411003:Experience

ZJU-China 2013 tests K411003

ZJU-China 2013 continued the work of 2012 team to test more parameters of this typical riboswitch part.

The protocol is as follows:

1. Cultivate the BL21 containing BBa_K411003 in LB at 37℃ 180rpm until OD600 reaches 0.5.

2. Induce the bacteria with 1mM IPTG (final concentration), and cultivate them at 37℃180rpm for 4hrs.

3. Dilute the suspension culture with LB to 0.1OD600 . Theophylline is added at concentration ranging from 1.00E-05 mol/L to1.00E-01 mol/L.

4. Measure the RFU ( ex488/em525) and OD600 synchronously.

5. Data Analysis

The RFU/OD decreases along the time which we believe the rapid growth of OD contributed the most.

This figure shows how the whole GFP Change Rate(GCR) changes at different time and under different concentration. Initially, the culture induced by 0.5M Theophylline has the largest whole GCR. And almost every culture’s GCR experiences decrement along time.

GFP Change Rate versus Concentration at 100 minutes after Theophylline was added.

ZJU-China 2012 tests K411003

The 8 different concentration of theophylline comparision on part K411003 theophylline robswitch tagged with GFP. Excitation at 480nm and emission at 535nm. Up to 10mM theophylline, cells didn’t show obvious side effects and GFP production is proportioned with theophylline concentration, showing that K411003 is an effective riboswitch which can be regulated by theophylline. When theophylline concentration is beyond a certain degree (about 10 mM), it somewhat affect cell growth and GFP production.

Results show that this is an effecitve riboswitch regulated by theophylline.

We improve this part, embed an RNA scaffold after the theophylline aptamer in K411001 in K411003 to become riboscaffold K738002, thus empower K411001 & K411003 with new application. We use split GFP to test how theophylline affect the 3D structure of whole RNA. [http://2012.igem.org/Team:ZJU-China/project.htm ZJU-China 2012 wiki]