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From 2012.igem.org

Preparation: Characterize parts in registry

 

We based our design partly on existing parts K537009 and K411003. These two parts are riboswitches sharing the same theophylline aptamer. We utilized their aptamer, embed an RNA scaffold after the theophylline aptamer in K539003 of K537009 and K411001 of K411003, to become alloscaffold K738002, thus empower K537003 & K537009 with new application. We mutated 8 bases of their aptamer to improve its compatibility with scaffold.

To make tentative steps in order to fix the theophylline concentration of our clover version coexpression experiment, we test these two theophylline riboswitches tagged with fluorescent proteins.

 

1. BioBrick Part K537009:

 

To characterise the theophylline riboswitches (part K537009, iGEM11_WITS_CSIR_SA), we quantified their activation at different theophylline concentrations (0 mM, 1 mM, 5 mM, 10 mM and 20 mM) over 2 hours using fluorometry.

 

Competent E. coli (strain DH5a) cells were transformed with plasmid vectors containing the "Promoter-Theophylline riboswitch -Venus-Double terminator". The bacterial colony appeal pink.

 

Cultured until the mid-log phase of growth, a different concentration of theophylline was added to each culture for induction. The activation of the riboswitch was detected as a fluorescent response as a result of increased translation of the fluorescent protein Venus, in the presence of the activator. Before fluorescence assay, we wash the culture with PBS.

 

A Synergy hybrid reader was used to excite the cultures at 505 nm and the intensity of the emission was detected at 535 nm. Empty bacteria were used to correct for auto fluorescence (IGEM11_WITS_CSIR_SA offered exciting at 514nm and emission at 528nm, but 514&528 is too close for our machine to detect.)

 

We have two end points of the OD 630 of each sample.

 

"Fluorescence intensity / OD" increases greatly with theophylline concentration.

 

Fig.12 The 5 different concentration of theophylline comparision on part K537009 theophylline robswitch tagged with venus YFP. Excitation at 505nm and emission at 535nm. Up to 20mM theophylline, cells didn't show side effects and YFP production is proportioned with theophylline concentration, showing that K537009 is an effective riboswitch which can be regulated by theophylline.

 

Fluorescence Microscope could also show this part work beautifully.

 

Except for the difference that though K537009 is an YFP, we excite it at 532nm (green light) and it glow red.

 

Fig.13 5 different concentration of theophylline comparision on part K537009 theophylline riboswitch tagged with venus YFP. The brightfield (BF) images in the right column depict all bacterial cells. The venus images in the left column depict bacterial cells which emitted fluorescence. We excite it at 532nm (green light) and it glow red, seeing the obvious trend that when adding more theophylline, cells showing Veuns appeal more.

 

2. BioBrick Part K411003:

 

This is a"pLAC promotor,Theophylline-inducible Riboswitch, GFP+Terminator" part made by 2010 NYMU-Taipe. We make similar tests as K537009 at different theophylline concentrations (0 mM, 0.1mM, 0.3mM, 0.5 mM,1mM,5mM,10mM and 20mM) over 2 hours using fluorometry.

 

Synergy hybrid reader detects effective effects of theophylline on GFP production.

 

 

Fig.14 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 the aptamer does have response to theophylline and it would be safe for us to utilize and improve.

Through Fig9 and Fig11 we find that when theophylline concentration scale is 0-1mM (especially 0-0.5mM); the response of fluorescence protein to theophylline is more significant with bigger slope. So we decided to carry out our alloscaffolds’ characterizations with theophylline concentration scale 0-1mM.