BASIL2023GVQ8DN35 - Revision history

Bonnie Hall at 03:20, 4 February 2024

Bonnie Hall — Sun, 04 Feb 2024 03:20:38 GMT

←Older revision		Revision as of 03:20, 4 February 2024
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	== Computational Structure ==		== Computational Structure ==

-	<scene name='95/957644/Q8dn35_structure/2'>Click to display 3D model of Q8DN35</scene>	+	<scene name='95/957644/Q8dn35_structure/2'>Click to display 3D model of Q8DN35</scene>

	== Introduction ==		== Introduction ==

Lucas Kramer at 14:14, 26 April 2023

Lucas Kramer — Wed, 26 Apr 2023 14:14:09 GMT

←Older revision		Revision as of 14:14, 26 April 2023
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	<scene name='95/957644/Q8dn35_structure/2'>Click to display 3D model of Q8DN35</scene>		<scene name='95/957644/Q8dn35_structure/2'>Click to display 3D model of Q8DN35</scene>
		+
		+	== Introduction ==
		+	Q8DN35 is an enzyme with a computational structure but no known function. Our enzyme was predicted to be a kinase. The hypothesized function of this protein was that of a glucokinase, and through in silico research, two substrates were selected for further testing. Fructose-6-Phosphate (F6P) and N-acetyl-D-glucosamine (NAG) were selected based on binding affinity during PyRx docking analysis and based on correct binding site location. NetGO machine learning algorithms predicted the function of Q8DN35 to be a glucokinase, with N-acetylglucokinase activity also predicted deeply. Kinetic activity was analyzed to determine whether F6P or NAG were correct substrates. Bradford standards and gel electrophoresis were also conducted to determine concentration of Q8DN35 and to ensure the protein was present.

	== Function ==		== Function ==

Lucas Kramer at 00:15, 25 April 2023

Lucas Kramer — Tue, 25 Apr 2023 00:15:05 GMT

←Older revision		Revision as of 00:15, 25 April 2023
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	[[Image:Q8DN35 DNA Gel.png]]		[[Image:Q8DN35 DNA Gel.png]]

-	~~The presence of two bands when using both restriction enzymes allows us to confirm presence of the Q8DN35 DNA as indicated by the plasmid map for our DNA.~~	+
	== Protein purification analysis ==		== Protein purification analysis ==
	Through the use of a gravity purification column and Ni-NTA resin, Q8DN35 was purified and a Bradford analysis was conducted to determine the amount of protein in the purified sample. This purification resulted in 0.055 mg/mL of protein being purified. Through SDS-PAGE analysis, observation of purified protein was hard to see in the fraction column but large banding occurred in the pre- and post-induction samples around 31.88 kDa, which is the predicted molecular weight for Q8DN35. Further analysis with more concentrated protein sample will need to be done to confirm correct protein positioning.		Through the use of a gravity purification column and Ni-NTA resin, Q8DN35 was purified and a Bradford analysis was conducted to determine the amount of protein in the purified sample. This purification resulted in 0.055 mg/mL of protein being purified. Through SDS-PAGE analysis, observation of purified protein was hard to see in the fraction column but large banding occurred in the pre- and post-induction samples around 31.88 kDa, which is the predicted molecular weight for Q8DN35. Further analysis with more concentrated protein sample will need to be done to confirm correct protein positioning.

Lucas Kramer at 00:14, 25 April 2023

Lucas Kramer — Tue, 25 Apr 2023 00:14:21 GMT

←Older revision		Revision as of 00:14, 25 April 2023
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	[[Image:Docking site Q8DN35.png]]		[[Image:Docking site Q8DN35.png]]

-	This image displays potential binding sites to the Q8DN35 protein.	+	This image displays potential binding sites to the Q8DN35 protein. Each grey "X" indicates a potential binding site. These binding sites were taken into consideration when looking at PyRx docking tools.

	== Experimental process ==		== Experimental process ==
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	== Q8DN35 DNA analysis ==		== Q8DN35 DNA analysis ==
-	To confirm presence of Q8DN35 DNA, plasmid digestion was performed to observe correct DNA.	+	To confirm presence of Q8DN35 DNA, plasmid digestion was performed to observe correct DNA. Sizes were determined from looking at the plasmid map provided for our kinase enzyme DNA. Cutting with both of the chosen restriction enzymes yielded a smaller band containing our insert and a larger band containing the rest of the plasmid. Using only one restriction enzyme gives one band with all of the plasmid DNA present.

	[[Image:Q8DN35 DNA Gel.png]]		[[Image:Q8DN35 DNA Gel.png]]
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	== References ==		== References ==
		+	1.Miyazono, K., Tabei, N., Morita, S., Ohnishi, Y., Horinouchi, S., & Tanokura, M. (2012). Substrate recognition mechanism and substrate-dependent conformational changes of an ROK family glucokinase from Streptomyces griseus. Journal of bacteriology, 194(3), 607–616. https://doi.org/10.1128/JB.06173-11
		+	2. The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.
		+
	<references/>		<references/>

Lucas Kramer at 19:33, 10 April 2023

Lucas Kramer — Mon, 10 Apr 2023 19:33:15 GMT

←Older revision		Revision as of 19:33, 10 April 2023
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	[[Image:Q8DN35 DNA Gel.png]]		[[Image:Q8DN35 DNA Gel.png]]

		+	The presence of two bands when using both restriction enzymes allows us to confirm presence of the Q8DN35 DNA as indicated by the plasmid map for our DNA.
	== Protein purification analysis ==		== Protein purification analysis ==
	Through the use of a gravity purification column and Ni-NTA resin, Q8DN35 was purified and a Bradford analysis was conducted to determine the amount of protein in the purified sample. This purification resulted in 0.055 mg/mL of protein being purified. Through SDS-PAGE analysis, observation of purified protein was hard to see in the fraction column but large banding occurred in the pre- and post-induction samples around 31.88 kDa, which is the predicted molecular weight for Q8DN35. Further analysis with more concentrated protein sample will need to be done to confirm correct protein positioning.		Through the use of a gravity purification column and Ni-NTA resin, Q8DN35 was purified and a Bradford analysis was conducted to determine the amount of protein in the purified sample. This purification resulted in 0.055 mg/mL of protein being purified. Through SDS-PAGE analysis, observation of purified protein was hard to see in the fraction column but large banding occurred in the pre- and post-induction samples around 31.88 kDa, which is the predicted molecular weight for Q8DN35. Further analysis with more concentrated protein sample will need to be done to confirm correct protein positioning.

Lucas Kramer at 18:19, 10 April 2023

Lucas Kramer — Mon, 10 Apr 2023 18:19:50 GMT

←Older revision		Revision as of 18:19, 10 April 2023
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	== Experimental process ==		== Experimental process ==

-	[[Image:Experimental procedure Q8DN35.png]]	+	[[Image:Experimental procedure Q8DN35 smaller.png]]

	== Q8DN35 DNA analysis ==		== Q8DN35 DNA analysis ==

Lucas Kramer at 18:17, 10 April 2023

Lucas Kramer — Mon, 10 Apr 2023 18:17:25 GMT

←Older revision		Revision as of 18:17, 10 April 2023
Line 18:		Line 18:

	== Experimental process ==		== Experimental process ==
		+
		+	[[Image:Experimental procedure Q8DN35.png]]

	== Q8DN35 DNA analysis ==		== Q8DN35 DNA analysis ==

Lucas Kramer at 18:16, 10 April 2023

Lucas Kramer — Mon, 10 Apr 2023 18:16:23 GMT

←Older revision		Revision as of 18:16, 10 April 2023
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	This image displays potential binding sites to the Q8DN35 protein.		This image displays potential binding sites to the Q8DN35 protein.
		+
		+	== Experimental process ==

	== Q8DN35 DNA analysis ==		== Q8DN35 DNA analysis ==

Lucas Kramer at 18:15, 10 April 2023

Lucas Kramer — Mon, 10 Apr 2023 18:15:15 GMT

←Older revision		Revision as of 18:15, 10 April 2023
Line 19:		Line 19:
	== Q8DN35 DNA analysis ==		== Q8DN35 DNA analysis ==
	To confirm presence of Q8DN35 DNA, plasmid digestion was performed to observe correct DNA.		To confirm presence of Q8DN35 DNA, plasmid digestion was performed to observe correct DNA.
		+
		+	[[Image:Q8DN35 DNA Gel.png]]

	== Protein purification analysis ==		== Protein purification analysis ==

Lucas Kramer at 18:13, 10 April 2023

Lucas Kramer — Mon, 10 Apr 2023 18:13:59 GMT

←Older revision		Revision as of 18:13, 10 April 2023
Line 16:		Line 16:

	This image displays potential binding sites to the Q8DN35 protein.		This image displays potential binding sites to the Q8DN35 protein.
		+
		+	== Q8DN35 DNA analysis ==
		+	To confirm presence of Q8DN35 DNA, plasmid digestion was performed to observe correct DNA.
		+
	== Protein purification analysis ==		== Protein purification analysis ==
	Through the use of a gravity purification column and Ni-NTA resin, Q8DN35 was purified and a Bradford analysis was conducted to determine the amount of protein in the purified sample. This purification resulted in 0.055 mg/mL of protein being purified. Through SDS-PAGE analysis, observation of purified protein was hard to see in the fraction column but large banding occurred in the pre- and post-induction samples around 31.88 kDa, which is the predicted molecular weight for Q8DN35. Further analysis with more concentrated protein sample will need to be done to confirm correct protein positioning.		Through the use of a gravity purification column and Ni-NTA resin, Q8DN35 was purified and a Bradford analysis was conducted to determine the amount of protein in the purified sample. This purification resulted in 0.055 mg/mL of protein being purified. Through SDS-PAGE analysis, observation of purified protein was hard to see in the fraction column but large banding occurred in the pre- and post-induction samples around 31.88 kDa, which is the predicted molecular weight for Q8DN35. Further analysis with more concentrated protein sample will need to be done to confirm correct protein positioning.