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Team:Nevada/Project
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==== '''''AGP/inv'' Operon''' ==== | ==== '''''AGP/inv'' Operon''' ==== | ||
| + | AGP knockout/inv operon | ||
| + | Construct Design: (Image): Marguerite, put the photo of the agp knockout/inv operon you made here | ||
| + | Features: | ||
| + | 1. AGP Knockout: AGP encodes for the enzyme ADP-glucose-pyrophosphorylase. This enzyme is involved in glycogen biosynthesis from glucose intermediates and catalyzes the following reaction: ATP + alpha-D-glucose 1-phosphate = diphosphate + ADP-glucose. Knocking out this gene in Synechocystis PCC 6803 will decrease the amount of glucose being used to produce glycogen chains. The excess glucose that accumulates as a result of knocking out AGP is, alternatively, converted into sucrose. AGP was isolated from Synechocystis PCC 6803 genomic DNA. | ||
| + | |||
| + | 2. Inv: inv encodes for the enzyme invertase which catalyzes the hydrolysis of sucrose into glucose and fructose through the following reaction: | ||
| + | |||
| + | (Insert AGP Image 1) | ||
| + | |||
| + | Thus, the increased quantity of sucrose resulting from the AGP knockout will be catabolized into free glucose and fructose. inv was synthesized with transcriptional optimization and ligated into pSB1C3. | ||
| + | 3. petBD+ RBS: Light inducible promoter and ribosome binding site. Allows transcription and translation of invertase in Synechocystis. Obtained from K390015 (Utah State). | ||
| + | |||
| + | 4. Kanamyacin resistance selection: Obtained from pUC4K (Stanford). | ||
| + | |||
| + | |||
| + | |||
| + | Approach: | ||
| + | Gibson Assembly: | ||
| + | 1. Primer Design: Forward and reverse primers for each DNA part were designed with 20 base pair overlapping sequences with the upstream and downstream flanking segments. This will create 40 base pair overlaps between neighboring parts in the construct. | ||
| + | 2. PCR: | ||
| + | (Insert AGP Image 2) | ||
| + | Inv, KnR, and petBD+RBS were amplified from pSB1C3, pUC4K (Stanford), and K390015 (Utah State) respectively under standard PCR conditions. | ||
| + | (Insert AGP Image 3_Table) | ||
| + | |||
| + | (Insert AGP Image 4) | ||
| + | AGP in pSB1A3 will be amplified to include the vector. This will be used as the backbone for Gibson Assembly. | ||
| + | |||
| + | 3.Assembly of Parts: The above parts will be assembled into the following construct: | ||
| + | |||
| + | (Insert AGP Image 5) | ||
| + | |||
| + | Transformation: Synechocystis will be naturally transformed via homologous recombination. | ||
| + | (Insert AGP Image 6) | ||
Revision as of 13:49, 6 September 2011
You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing.
Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs)
Contents |
Introduction
Abstract:
Introduction:
Approach
E. coli
Fatty Acid Production
Ethanol Production
-Engineered Pyruvate Decarboxylase and Alcohol Dehydrogenase coding regions (based on Zymomonas mobilis, a microorganism that naturally produces ethanol).
- Removed PDC/ADH from pUC57 vector and put into pSB1C3 -Sequenced and confirmed PDC/ADH in pSB1C3 -Submit PDC/ADH/pSB1C3 to iGEM
-Put a constitutive promoter (σ70) in front of PDC/ADH genes to test expression of ethanol. -Opened up σ70 in Amp vector, then ligated with PDC/ADH. -Sequenced and confirmed σ70/PDC/ADH in Amp vector.
With σ70/PDC/ADH in Amp vector (in NEB 10β cells): -Performed Ethanol Detection test- no samples produced detectable ethanol above background… -Performed ADH Enzymatic test- no ADH detected in assay.
-created “Aldehyde Detection Plates” to test PDC presence & functionality -re-transformed σ70/PDC/ADH in Amp vector into NEB Iq cells -Plates & colonies turn Bright pink- so Aldehydes ARE present (possibly being secreted)
-When ethanol can be detected from σ70/PDC/ADH in Amp vector, I will get -σ70/PDC/ADH into pSB1C3 and submit as an iGEM part. -We are still working on getting pTRC in front of PDC/ADH in pSB1C3
E. coli Results
Cyanobacteria
The cyanobacteria will take up atmospheric CO2 and fix the carbon. From this carbon source sucrose is synthesized, and invertase converts it into glucose and fructose, which is finally transported out of the cell by glF.
AGP/inv Operon
AGP knockout/inv operon Construct Design: (Image): Marguerite, put the photo of the agp knockout/inv operon you made here Features: 1. AGP Knockout: AGP encodes for the enzyme ADP-glucose-pyrophosphorylase. This enzyme is involved in glycogen biosynthesis from glucose intermediates and catalyzes the following reaction: ATP + alpha-D-glucose 1-phosphate = diphosphate + ADP-glucose. Knocking out this gene in Synechocystis PCC 6803 will decrease the amount of glucose being used to produce glycogen chains. The excess glucose that accumulates as a result of knocking out AGP is, alternatively, converted into sucrose. AGP was isolated from Synechocystis PCC 6803 genomic DNA.
2. Inv: inv encodes for the enzyme invertase which catalyzes the hydrolysis of sucrose into glucose and fructose through the following reaction:
(Insert AGP Image 1)
Thus, the increased quantity of sucrose resulting from the AGP knockout will be catabolized into free glucose and fructose. inv was synthesized with transcriptional optimization and ligated into pSB1C3. 3. petBD+ RBS: Light inducible promoter and ribosome binding site. Allows transcription and translation of invertase in Synechocystis. Obtained from K390015 (Utah State).
4. Kanamyacin resistance selection: Obtained from pUC4K (Stanford).
Approach: Gibson Assembly: 1. Primer Design: Forward and reverse primers for each DNA part were designed with 20 base pair overlapping sequences with the upstream and downstream flanking segments. This will create 40 base pair overlaps between neighboring parts in the construct. 2. PCR:
(Insert AGP Image 2)
Inv, KnR, and petBD+RBS were amplified from pSB1C3, pUC4K (Stanford), and K390015 (Utah State) respectively under standard PCR conditions. (Insert AGP Image 3_Table)
(Insert AGP Image 4) AGP in pSB1A3 will be amplified to include the vector. This will be used as the backbone for Gibson Assembly.
3.Assembly of Parts: The above parts will be assembled into the following construct:
(Insert AGP Image 5)
Transformation: Synechocystis will be naturally transformed via homologous recombination. (Insert AGP Image 6)
