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	<img src="https://static.igem.org/mediawiki/2012/b/bb/WT_PUF-PIN%2C_6-2%2C7-2_PUF-PIN%2C_WT_PUF-aGFP_Lysates.png" height=85% width=85%></center>		<img src="https://static.igem.org/mediawiki/2012/b/bb/WT_PUF-PIN%2C_6-2%2C7-2_PUF-PIN%2C_WT_PUF-aGFP_Lysates.png" height=85% width=85%></center>
-	<br/><b>Fig 1. 1 L culture incubated at 37oC till 0.5 nm optical density after inoculation with 5 mL of overnight for WT PUF-PIN and 6-2/7-2 PUF-PIN. 2 mM IPTG induction for 2 hours. 200 mL cultures incubated at 37oC till 1 nm optical density after inoculation with 2 mL of overnight for WT PUF-αGFP cultures. 2 mM IPTG induction for various hours. </b>	+	<br/><b>Fig 1.</b> 1 L culture incubated at 37oC till 0.5 nm optical density after inoculation with 5 mL of overnight for WT PUF-PIN and 6-2/7-2 PUF-PIN. 2 mM IPTG induction for 2 hours. 200 mL cultures incubated at 37oC till 1 nm optical density after inoculation with 2 mL of overnight for WT PUF-αGFP cultures. 2 mM IPTG induction for various hours.
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	<img src="https://static.igem.org/mediawiki/2012/4/41/WT_PUF-PIN_Protein_Purification.png" height=85% width=85%></center>		<img src="https://static.igem.org/mediawiki/2012/4/41/WT_PUF-PIN_Protein_Purification.png" height=85% width=85%></center>
-	<br/><b>Fig 2.</b>	+	<br/><b>Fig 2.</b> 1 L culture incubated at 37oC till 0.5 nm optical density after inoculation with 5 mL of overnight. 2 mM IPTG induction for 2 hours. His-Tag Ni-NTA purification, centrifuged with Millipore 30kDa cutoff ultracentrifuge tubes.
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	<img src="https://static.igem.org/mediawiki/2012/6/6f/6-2%2C7-2_PUF-PIN_Protein_Purification.png" height=85% width=85%></center>		<img src="https://static.igem.org/mediawiki/2012/6/6f/6-2%2C7-2_PUF-PIN_Protein_Purification.png" height=85% width=85%></center>
-	<br/><b>Fig 3.</b>	+	<br/><b>Fig 3.</b> 1 L culture incubated at 37oC till 0.5 nm optical density after inoculation with 5 mL of overnight. 2 mM IPTG induction for 2 hours. His-Tag Ni-NTA purification, centrifuged with Millipore 30kDa cutoff ultracentrifuge tubes.
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Revision as of 23:20, 30 September 2012

RNA Scaffold Overview

In order to provide a direct application for the RNA binding abilities of PUF, an RNA scaffold was designed with the idea of serving as a platform for an enzyme conveyor belt. The array of enzymatic pathways which could be enhanced by a scaffold are numerous, though, we projected to increase efficiency and production of a resveratrol derivative called piceatannol.

The project consists of a couple parts, each a proof of concept and build-up of previous ones. The start of the project consisted of designing an RNA scaffold which is best tailored to PUF binding in a spatially specific manner. Once the scaffold was designed, synthesized, and purified it was important to show not only that PUF can bind specifically to its designated sites, but that the scaffold can support a concept such as a biological conveyor belt.

One assay which was designed to prove this was incubation of the RNA scaffold with non-specific endonucleases bound to PUF. The length of digested RNA parts would prove that PUF was binding specifically and appropriately to the designated sequences. Another assay would include tethering a split-fluorescent protein to wild-type and mutant PUF. An in-vitro gel-shift assay, or EMSA, would once again prove that PUF is binding the the appropriate sites. More importantly, an in-vivo experiment which shows fluorescence with presence of the scaffold and darkness without the scaffold would prove efficient enzymatic pathways could be achieved.

RNA Scaffold Design

RNA Scaffold Data

Fig 1. 1 L culture incubated at 37oC till 0.5 nm optical density after inoculation with 5 mL of overnight for WT PUF-PIN and 6-2/7-2 PUF-PIN. 2 mM IPTG induction for 2 hours. 200 mL cultures incubated at 37oC till 1 nm optical density after inoculation with 2 mL of overnight for WT PUF-αGFP cultures. 2 mM IPTG induction for various hours.


Fig 2. 1 L culture incubated at 37oC till 0.5 nm optical density after inoculation with 5 mL of overnight. 2 mM IPTG induction for 2 hours. His-Tag Ni-NTA purification, centrifuged with Millipore 30kDa cutoff ultracentrifuge tubes.


Fig 3. 1 L culture incubated at 37oC till 0.5 nm optical density after inoculation with 5 mL of overnight. 2 mM IPTG induction for 2 hours. His-Tag Ni-NTA purification, centrifuged with Millipore 30kDa cutoff ultracentrifuge tubes.

In-Vitro Transcription with MEGAscript® T7 Kit (Invitrogen)

Fig 4.

PUF Tether Design

PUF Tethering Data

Fig 1.


Fig 2.


Fig 3.


Fig 4.


Fig 5.

Conclusion