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THECSiEGENE

by Timothy Lane

Timothy Lane CIS 1020 Final Project

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Overview

Introduction to the CSiE Gene Control of Gene Expression Uses for CSiE Proposed Experiments Proposed ResultsConclusion

Timothy Lane CIS 1020 Final Project

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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

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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

The Bacterial Life Cycle

Timothy Lane CIS 1020 Final Project

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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)

THECSiEGENE

Timothy Lane CIS 1020 Final Project

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Control of Gene Expression

Regulation of CSiE can be assisted by:Sigma S – Primary stress regulatorSigma 70 – Housekeeping factorcAMP-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

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• Lowered cellular metabolic rates• Less cell wall permeability

• Adaptation to nutrient limitation• Survival of population density• Ensuring conservation of energy

within the cell

Uses of CSiE

The effects of forcing cells into their stationary phase result in:

Timothy Lane CIS 1020 Final Project

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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

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How Transcription Works

Timothy Lane CIS 1020 Final Project

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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

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Proposed Results Using 10X Starting Templates

Fluorescence Threshold OutputCycle Threshold Analysis

Cells maintained log phase for a longer duration

Timothy Lane CIS 1020 Final Project

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Proposed Cycle Threshold Results Analysis

Cells reformed back into log phase with accelerated growth

BTEC 2040: Advanced MolecularMethods - Dr. Jean M. Bower

Tube Strain Reverse Transcriptase

Gene Cycle Threshold

1 Wild Type Positive PG50 15.97

2 Wild Type Positive CSiE 12.34

3 Wild Type Negative PG50 -

4 Wild Type Negative CSiE -

5 Re-Eng Positive PG50 15.85

6 Re-Eng Positive CSiE 24.68

7 Re-Eng Negative PG50 -

8 Re-Eng Negative CSiE -

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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 PG50No Ct value was shown for the –ve Reverse

Transcriptase, indicating purity of samples.

Timothy Lane CIS 1020 Final Project

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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