BME494 Sp2014 Dhatt: Difference between revisions
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'''TYPE IIS ASSEMBLY''' | '''TYPE IIS ASSEMBLY''' | ||
'''PCR''' | '''PCR'''<br> | ||
Polymerase Chain Reaction (PCR) will be used for amplification of the DNA parts. PCR is the process of adding DNA, primers, nucleotides and DNA polymerase to a tube which produces system through assembly pot process after placed in a thermocycler and placed through many cycles. PCR consists of three specific temperature steps: denaturation, annealing, and elongation. Denaturation allows the DNA template to be heated to a specific temperature and yield single-stranded DNA molecules. The reaction temperature is then lowered for a short period of time in order to allow the complementary DNA primers to anneal to the single-stranded DNA template. The primers are then elongated by the DNA polymerase that is used. The DNA polymerase then allows for the cycle to be repeated multiple times resulting in thousands of copies of the desired fragments. | Polymerase Chain Reaction (PCR) will be used for amplification of the DNA parts. PCR is the process of adding DNA, primers, nucleotides and DNA polymerase to a tube which produces system through assembly pot process after placed in a thermocycler and placed through many cycles. PCR consists of three specific temperature steps: denaturation, annealing, and elongation. Denaturation allows the DNA template to be heated to a specific temperature and yield single-stranded DNA molecules. The reaction temperature is then lowered for a short period of time in order to allow the complementary DNA primers to anneal to the single-stranded DNA template. The primers are then elongated by the DNA polymerase that is used. The DNA polymerase then allows for the cycle to be repeated multiple times resulting in thousands of copies of the desired fragments. | ||
'''Digestion/Ligation Reaction''' | '''Digestion/Ligation Reaction'''<br> | ||
The digestion/ligation reaction allows for dilution the purified PCR products to volume of 20 ul. The digestion/ligation reaction uses BsmB1, a Type II restriction enzyme, which cuts DNA fragments to create complementary overhangs (sticky ends). These sticky ends anneal via DNA base pairs. Primers introduce these complementary overhangs to the DNA. T4 ligase is used to seal the gaps between base pairs and create a finished system. | The digestion/ligation reaction allows for dilution the purified PCR products to volume of 20 ul. The digestion/ligation reaction uses BsmB1, a Type II restriction enzyme, which cuts DNA fragments to create complementary overhangs (sticky ends). These sticky ends anneal via DNA base pairs. Primers introduce these complementary overhangs to the DNA. T4 ligase is used to seal the gaps between base pairs and create a finished system. | ||
Revision as of 15:31, 8 May 2014
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Background & Proposed ApplicationBACKGROUND
The synthetic system modeled in the “Construction of a genetic toggle switch In Escherichia coli” uses two different states, an “on” state” and an “off” state (Gardner et all 2000). The classical system uses two repressor and two promoter pairs in which both repressors are inhibited by a different inducer, aTc and IPTG. The promoters were placed next to the repressor gene of the opposite pair. This system represents a bistable gene-regulatory network where only one promoter can be expressed at one time because the expression of one promoter repressed expression of the other promoter. The on-state of the cell was represented by placing a green fluorescent protein (GFP) transcription gene downstream of the on-state promoter.
Globally, 227-285 million individuals have diabetes and about 90% of these individuals have Type II diabetes. In 2011, 1.4 million deaths occurred worldwide due to the result of diabetes making it the 8th leading cause of death. This number is estimated to double in the next 15 years, therefore, there is a need for a rapid diagnostic test.
Design of a New DeviceThe device will be designed as a diagnostic tool for diabetes and the functionality of the genetic switch replicates that of an “AND” logic gate. The device will require two conditions to be true in order for an output to be produced. One condition is that IPTG must be present in the device’s environment. When IPTG is present, it will bind to the LacI repressor and allow for transcription to continue. The other condition is that glucose levels in the device’s environment must be low. Glucose levels affect production of cAMP inversely; when glucose levels are high, cAMP production decreases and when glucose levels are low, cAMP production increases. cAMP binds to catabolite activator protein (CAP_ to form the CAP-cAMP complex. For the complex, cAMP must be present and glucose levels must be low. This complex is the required input of the device. In the natural lac operon, the CAP-cAMP complex leads to activation of gene expression from the lac operon. If glucose is present, cAMP levels will be low and the host will metabolize glucose if lactose is present.
Building the New DeviceSYNTHETIC DNA LAYOUT Type IIs Assembly was used to build this lac switch. Type IIs Assembly allows for parts to be assembled in one step. For Type IIs Assembly, forward and reverse primers are needed to be created and placed in the system in order to create sticky ends that can bind various parts together. To put the pieces together, PCR is implemented, which allows all the parts to be replicated thousands of times in order to produce a desired final product. Digestion and ligation is used during which BsmBI cuts the DNA fragments and creates complementary overhangs that anneal via base pairing.
RESOURCES The following BioBrick parts, found on the iGEM registry website, will be used to build the new device: K418003 - composite Lac promoter inducible by IPTG • Size: 1416 base pairs • LacI present: transcription inhibited • LacI absent: transcription promoted • LacI inhibited by IPTG B0030 – Strong RBS • Size: 15 base pairs K259006 – Composite part made from green fluorescent protein (GFP) and double terminator • IPTG present: GFP produced • IPTG absent: GFP not produced, clear solution • Size: 823 base pairs pSB1A3 – BioBrick assembly plasmid • Size:2155 base pairs
PCR Polymerase Chain Reaction (PCR) will be used for amplification of the DNA parts. PCR is the process of adding DNA, primers, nucleotides and DNA polymerase to a tube which produces system through assembly pot process after placed in a thermocycler and placed through many cycles. PCR consists of three specific temperature steps: denaturation, annealing, and elongation. Denaturation allows the DNA template to be heated to a specific temperature and yield single-stranded DNA molecules. The reaction temperature is then lowered for a short period of time in order to allow the complementary DNA primers to anneal to the single-stranded DNA template. The primers are then elongated by the DNA polymerase that is used. The DNA polymerase then allows for the cycle to be repeated multiple times resulting in thousands of copies of the desired fragments.
Forward Primer Composite Lac Promoter: 5'-cacaccaCGTCTCatagattgacagctagctca Reverse Primer Composite Lac Promoter: 5'-cacaccaCGTCTCatgtgtgtgctcagtatctt Forward Primer Operator: 5'-cacaccaCGTCTCacaatacgcaaaccgc Reverse Primer Operator: 5'-cacaccaCGTCTCatttctgtgtgaaattgtta Forward Primer RBS: 5'-cacaccaCGTCTCattaaagaggagaaa Reverse Primer RBS: 5'-cacaccaCGTCTCaaattttctcctctttaat Forward Primer GFP with terminator: 5'-cacaccaCGTCTCaatgcgtaaaggagaa Reverse Primer GFP with terminator: 5'-cacaccaCGTCTCatagtaaataataaaaaagc Forward Primer for the mutation: 5’-agctgttgccGgtctcactgg Reverse Primer for the mutation: 5’-ccagtgagacCggcaacagct
The PCR reagents used the following thermal cycling properties:
The Digestion/Ligation reagents used the following thermal cycling properties:
Testing the New DeviceLAC OPERON MODEL SIMULATION
Similarities:
Differences:
Human Practices
About the Designer
References[1] Full reference. [2] Full reference. [3] Full reference.
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