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The CSiE Gene

The CSiE Gene. by Timothy Lane. Timothy Lane CIS 1020 Final Project. Overview. Introduction to the CSiE Gene Control of Gene Expression Uses for CSiE Proposed Experiments Proposed Results Conclusion. InTroduction.

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The CSiE Gene

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  1. TheCSiEGene by Timothy Lane Timothy Lane CIS 1020 Final Project

  2. Overview • Introduction to the CSiE Gene • Control of Gene Expression • Uses for CSiE • Proposed Experiments • Proposed Results • Conclusion Timothy Lane CIS 1020 Final Project

  3. InTroduction E.coli bacterial cells have 4,290 genes - These genes operate through the life cycle of the cell Timothy Lane CIS 1020 Final Project

  4. The Bacterial Life Cycle Bacterial lifecycle can be described in phases: • Lag phase - the adaptation delay before growth • Exponential growth or log phase • Stationary or Linear phase – where nutrients are limited • Decline or mortality phase - in which cells break down Timothy Lane CIS 1020 Final Project

  5. TheCSiEGene Of the 4,290 genes found in bacteria, only 115 genes are directly linked to initiating the stationary phase The CSiE Gene is one of the 115 genes associated with sending a cell into stationary phase: Part of the lifecycle where a population of cells have no net increase or decrease The CSiE Genehelps to decrease expression of the 115 genes by: Reducing RNA levels & limiting absorption and use of nutrients known as carbon starvation The CSiE Gene is a stationary phase-inducible gene under the control of: Sigma S &cAMP-CRP complex (global regulatory factors) Timothy Lane CIS 1020 Final Project

  6. Control of Gene Expression Regulation of CSiE can be assisted by: Sigma S – Primary stress regulator Sigma 70 – Housekeeping factor cAMP-CRP – Secondary regulator Transcription is initiated by: Sigma S at base 33 of the CSiE’spromoter initiation codon If Base 72 upstream of the CSiE initiation codon is deleted, CSiE transcription is controlled by cAMP-CRP alone If Base -38 upstream of the start codon is deleted, CSiE transcription is controlled purely by cAMP-CRP BTEC 2040: Advanced MolecularMethods - Dr. Jean M. Bower

  7. Uses of CSiE • Lowered cellular metabolic rates • Less cell wall permeability • Adaptation to nutrient limitation • Survival of population density • Ensuring conservation of energy within the cell The effects of forcing cells into their stationary phase result in: Timothy Lane CIS 1020 Final Project

  8. Proposed Experiment Transform MG1655 e.coli by heatshock, and insert the pKD3 plasmid Knock out rPOS gene Purify the PCR product with QiagenRNeasy mini kit Insert CSiE primers and the Reverse transcriptase, ‘Superscript’ Amplify by Real time PCR Run a melt curve to verify amplicons Reverse engineer the CSiE gene into pKD3, with appropriate 5’-3’ to 3’-5’ primers Once viability is established amplify pKD3 using RT-qPCR Timothy Lane CIS 1020 Final Project

  9. How Transcription Works Timothy Lane CIS 1020 Final Project

  10. Melting Curve Analysis Melt curve showing single amplicon peak of 87.5 °C Denaturization occurs in one temperature zone, suggesting purity of rRNA Timothy Lane CIS 1020 Final Project

  11. Proposed Results Using 10X Starting Templates Cycle Threshold Analysis Fluorescence Threshold Output Cells maintained log phase for a longer duration Timothy Lane CIS 1020 Final Project

  12. Proposed Cycle Threshold Results Analysis Cells reformed back into log phase with accelerated growth BTEC 2040: Advanced MolecularMethods - Dr. Jean M. Bower

  13. CSiE Difference = Re-Engineered / Wild Type = 24.68 / 12.34 = 2 Fold Difference CsiE Gene vs. PG50 Loading Control Results PG50 Difference = Re-Engineered / Wild Type = 15.85 / 15.97 = 0.992 ≈ 1 Fold Difference • Expression of CSiE gene was double that of PG50 • No Ct value was shown for the –ve Reverse Transcriptase, indicating purity of samples. Timothy Lane CIS 1020 Final Project

  14. Re-engineered MG1655 e.coli bacterial cells, with the reversed CSiE gene, showed a longer duration of exponential growth. Conclusion The limitations of this experiment were that they were carried out in bacterial cells only, however there is no evidence of success in eukaryotic cells at the moment. The future of cells with the re-engineered CSiE gene could show significant cellular lifecycle improvement, and could be used in cell rejuvenation. Timothy Lane CIS 1020 Final Project

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