TheCSiEGene by Timothy Lane Timothy Lane CIS 1020 Final Project

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

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

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

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

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

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

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

How Transcription Works Timothy Lane CIS 1020 Final Project

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

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

Proposed Cycle Threshold Results Analysis Tube Strain Reverse Transcriptase Gene Cycle Threshold 1 Wild Type Positive PG50 15.97 2 CSiE 12.34 3 Negative - 4 5 Re-Eng 15.85 6 24.68 7 8 Cells reformed back into log phase with accelerated growth BTEC 2040: Advanced MolecularMethods - Dr. Jean M. Bower

CsiE Gene vs. PG50 Loading Control Results CSiE Difference = Re-Engineered / Wild Type = 24.68 / 12.34 = 2 Fold Difference 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

Conclusion Re-engineered MG1655 e.coli bacterial cells, with the reversed CSiE gene, showed a longer duration of exponential growth. 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