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	<center><h2>Conclusion</h2></center>		<center><h2>Conclusion</h2></center>
	<br/>		<br/>
		+
		+	PUF Tethering Conclusion:<br/><br/>
		+	After trying to optimize and tether PUF to cCFP several times, it was concluded that the tethering primers need to be changed. In Fig. 4 amplification of both PUF and cCFP was attempted before actual tethering and the result seen was primer dimers. Even after a gradient of temperatures, different PCR buffers, and primer concentrations were used, the result stayed the same. This showed that the problem laid in the primer sequences themselves.<br/><br/>
		+	Due to the difficulty of tethering two proteins together through a linker sequence and a shortage of time, it was decided upon that PUF (WT & 6-2/7-2) bound to split-fluorescent proteins would be sent for synthesis by a company. Split-GFP tethered to the PUF proteins was sent for GenScript to synthesize, however, only one construct was successfully produced. The production of the second construct was halted after numerous tries and thus, planned in vivo and in vitro tests were omitted due to the lack of both parts.
		+	<br/><br/>
		+
		+	RNA Scaffold Data:<br/><br/>
		+	In order to start the endonuclease assay mentioned in the design portion, a few necessary components needed to be collected. Both WT & 6-2/7-2 PUF-PIN were purified by Ni-NTA columns and then quantified through BCA analysis. Although the BCA analysis gave quantitative numbers, the SDS-PAGE gel showed large amounts of impurities in the elution fractions. Therefore, the concentrations determined by the BCA analysis should be taken as approximate due to the actual purity of the desired proteins being roughly 30-40%. Even though the purity of the proteins is sub-optimal, the RNA endonuclease enzymes used have multiple turnover, meaning they could cleave multiple RNA strands rather than cleaving one and ending the reaction. Before the endonuclease assay could be ran the RNA scaffold designed needed to be synthesized which was done through in-vitro transcription with the Invitrogen MEGAscript® T7 Kit. The expected length of the scaffold is 126bp and the NEB ssRNA ladder showed homology with the 150bp band. However, with presence of only one band and approximate length homology, it can be concluded that the expected scaffold construct was produced.
		+
		+	<br/><br/>
		+
		+	The preliminary endonuclease assay in Fig. 6 showed promising results. Although there are many variables which need to be optimized, there was cleavage of the scaffold seen most distinctly in wells with WT PUF-PIN and when both proteins, WT & 6-2/7-2, were present. There does not seem to be much activity of the 6-2/7-2 PUF-PIN protein (as seen in well 3 & 7), but that could be attributed to the fact that WT PUF-PIN was present in 6 fold greater concentration. The high background and speckles across the gel were most likely due to fiber particles falling off the paper towel used to clean the glass slides for casting the gel. It also seems that, contrary to literature, MgCl2 proved to work better than MnCl2 as ions for the endonuclease due to 6-2/7-2 PUF-PIN showing better cleavage in lane 7 than lane 3. Further optimized endonuclease assays with equal protein concentrations and MgCl2 will be ran to prove PUF binding specificity due to the present data not allowing for such a conclusive statement to be made.
		+
		+	<br/><br/>
		+
	</div>		</div>
	</div>		</div>

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Revision as of 03:25, 4 October 2012

RNA Scaffold Overview

The results covered in this section are of the experiments overviewed in the RNA Scaffold Design section.

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. SDS-PAGE 6% native gel stained with Coomassie Brilliant Blue for 1 hr. and destained with H20 for 1 hr.



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. SDS-PAGE 6% native gel stained with Coomassie Brilliant Blue for 1 hr. and destained with H20 for 1 hr.


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. SDS-PAGE 6% native gel stained with Coomassie Brilliant Blue for 1 hr. and destained with H20 for 1 hr.




Fig. 4. BCA analysis of WT & 6-2/7-2 PUF-PIN proteins to determine concentration (uM). 2 mg/mL BSA concentration used. Click on Fig. 4. to view it in a higher resolution.



Fig. 5. 10 well 10% 1mm urea denaturing acrylamide gel, post 2μg/mL EtBr staining for 20 min., destaining for 20 min. 1X TBE buffer, 120V for 55 min. In-Vitro Transcription with MEGAscript® T7 Kit (Invitrogen)



Fig. 6. 10 well 10% 1mm urea denaturing acrylamide gel, post 2μg/mL EtBr staining for 20 min., destaining for 20 min. 1X TBE buffer, 120V for 55 min. First 4 wells include 1 μL of 30 mM MnCl2 ions, last 4 wells include 1 μL of 30 mM MgCl2 ions. RNA samples were denatured for 3 min. at 95oC and then let to fold at 4oC for 5 min. Addition of 1 μL of annealing buffer (EDTA/Tris) before addition of protein. 30 min. incubation time with protein at 37oC and then addition of 2X 80% formamide/EDTA to stop the reaction. 66 nM concentration of RNA, 305 nM concentration of WT PUF-PIN, and 45 nM concentration of 6-2/7-2 PUF-PIN used.

PUF Tethering Data

Fig 1. 50mL 6 well 1% agarose gel with 5 μL of 10 mg/mL EtBr, 1X TAE buffer, 130V for 25 min. PCR amplifications done in GC Buffer, 68.7oC 30 sec. annealing time, 35 cycles for amplification and 10 cycles for tethering. PUF and cCFP (lanes 2 & 6) were first amplified and then used in the tethering reaction in lane 3.



Fig 2. 50mL 6 well 1% agarose gel with 5 μL of 10 mg/mL EtBr, 1X TAE buffer, 130V for 25 min. PCR amplifications done in HF Buffer, 68.7oC 30 sec. annealing time, 10 cycles of PCR.



Fig 3. 50mL 10 well 1% agarose gel with 5 μL of 10 mg/mL EtBr, 1X TAE buffer, 130V for 25 min. PCR amplifications done in GC Buffer, 1 min. annealing time, 10 cycles of PCR, half the volume of primers used (0.5 μL instead of 1 μL).



Fig 4. 50mL 6 well 1% agarose gel with 5 μL of 10 mg/mL EtBr, 1X TAE buffer, 130V for 25 min. PCR amplifications done in GC Buffer, 68.7oC 30 sec. annealing time, 25 cycles of PCR.

Conclusion

PUF Tethering Conclusion:

After trying to optimize and tether PUF to cCFP several times, it was concluded that the tethering primers need to be changed. In Fig. 4 amplification of both PUF and cCFP was attempted before actual tethering and the result seen was primer dimers. Even after a gradient of temperatures, different PCR buffers, and primer concentrations were used, the result stayed the same. This showed that the problem laid in the primer sequences themselves.

Due to the difficulty of tethering two proteins together through a linker sequence and a shortage of time, it was decided upon that PUF (WT & 6-2/7-2) bound to split-fluorescent proteins would be sent for synthesis by a company. Split-GFP tethered to the PUF proteins was sent for GenScript to synthesize, however, only one construct was successfully produced. The production of the second construct was halted after numerous tries and thus, planned in vivo and in vitro tests were omitted due to the lack of both parts.

RNA Scaffold Data:

In order to start the endonuclease assay mentioned in the design portion, a few necessary components needed to be collected. Both WT & 6-2/7-2 PUF-PIN were purified by Ni-NTA columns and then quantified through BCA analysis. Although the BCA analysis gave quantitative numbers, the SDS-PAGE gel showed large amounts of impurities in the elution fractions. Therefore, the concentrations determined by the BCA analysis should be taken as approximate due to the actual purity of the desired proteins being roughly 30-40%. Even though the purity of the proteins is sub-optimal, the RNA endonuclease enzymes used have multiple turnover, meaning they could cleave multiple RNA strands rather than cleaving one and ending the reaction. Before the endonuclease assay could be ran the RNA scaffold designed needed to be synthesized which was done through in-vitro transcription with the Invitrogen MEGAscript® T7 Kit. The expected length of the scaffold is 126bp and the NEB ssRNA ladder showed homology with the 150bp band. However, with presence of only one band and approximate length homology, it can be concluded that the expected scaffold construct was produced.

The preliminary endonuclease assay in Fig. 6 showed promising results. Although there are many variables which need to be optimized, there was cleavage of the scaffold seen most distinctly in wells with WT PUF-PIN and when both proteins, WT & 6-2/7-2, were present. There does not seem to be much activity of the 6-2/7-2 PUF-PIN protein (as seen in well 3 & 7), but that could be attributed to the fact that WT PUF-PIN was present in 6 fold greater concentration. The high background and speckles across the gel were most likely due to fiber particles falling off the paper towel used to clean the glass slides for casting the gel. It also seems that, contrary to literature, MgCl2 proved to work better than MnCl2 as ions for the endonuclease due to 6-2/7-2 PUF-PIN showing better cleavage in lane 7 than lane 3. Further optimized endonuclease assays with equal protein concentrations and MgCl2 will be ran to prove PUF binding specificity due to the present data not allowing for such a conclusive statement to be made.