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

2. 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

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
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

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
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

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