Nielson, Chris

Creation of Twelve Member Plasmid Library for Promoter Swapping to Control Chromosomal Gene Expression in E. Coli

Mentor: William R. McCleary, Department of Microbiology and Molecular Biology

Introduction

Metabolic engineering is becoming a very important area of research, allowing researchers to harness metabolic pathways to either eliminate or synthesize desired compounds. Understanding metabolic pathways by altering expression of proteins involved in the pathway helps to uncover thermodynamic bottlenecks which render certain pathways inefficient or infeasible. Altering expression of genes through the use of promoter swapping is a useful research technique used to understand important metabolic and physiologic pathways. Engineered promoters contained in plasmids can be inserted into chromosomal DNA through the utilization of promoter swapping to alter the expression of a given gene in order to better understand the pathway. We proposed creating a library of twelve plasmids containing promoter sequences capable of altering gene expression at varying, constitutive levels with an accompanying protocol for insertion of the engineered promoter sequence into the chromosome upstream of any gene of Escherichia coli. We have successfully synthesized two promoters which have altered β–galactosidase expression in cultured cells as desired.

Methodology

A. Cloning of pKE2 with insert to create template plasmid. Engineered plasmids were created by using pKE2 as a template plasmid. This plasmid contains genes conferring kanamycin resistance (Kmr) flanked by FRT recombination sites, and restriction endonuclease sites. Mutated promoter sequences were synthesized and ordered from Invitrogen Life Technologies and ligated into the template plasmid vector. Recombinant plasmids were transformed into BW25141 strain of E. coli and the cells were grown on media containing kanamycin to select for transformed cells. Plasmids were isolated and sequenced to confirm successful ligation. Successfully cloned plasmids were named pMP1-2 and pMP1-3.

B. Creation of PCR product from template plasmid pMP1 for electroporation into MG1655. These engineered plasmids were used as templates to create PCR products of our desired insert containing our promoter, Kmr genes, and up- and downstream homology sequences for insertion into the chromosome. Homology regions were added to the PCR product via primers with 5’ 20-nt overhang sequences homologous to the lacZ gene. PCR products were electroporated into MG1655 strain of E. coli containing the temperature sensitive plasmid, pKD46 which encodes for phage λ Red recombinase proteins. Cells containing our PCR product were selected for by growing them on plates containing kanamycin at 30° C. Selected cells were then grown at 42° C to eliminate pKD46. Whole colony PCR using lacfor and kan1 promoters was used to verify that kanamycin resistance was due to successful insertion of the promoter into the chromosome.

B. Creation of PCR product from template plasmid pMP1 for electroporation into MG1655. These engineered plasmids were used as templates to create PCR products of our desired insert containing our promoter, Kmr genes, and up- and downstream homology sequences for insertion into the chromosome. Homology regions were added to the PCR product via primers with 5’ 20-nt overhang sequences homologous to the lacZ gene. PCR products were electroporated into MG1655 strain of E. coli containing the temperature sensitive plasmid, pKD46 which encodes for phage λ Red recombinase proteins. Cells containing our PCR product were selected for by growing them on plates containing kanamycin at 30° C. Selected cells were then grown at 42° C to eliminate pKD46. Whole colony PCR using lacfor and kan1 promoters was used to verify that kanamycin resistance was due to successful insertion of the promoter into the chromosome.

C. Removal of the kanamycin resistance gene via transformation with pCP20. Selected cells were transformed using pCP20, a temperature sensitive plasmid containing ampicillin resistance genes and FLP recombinase genes in order to remove the Kmr cassette from the newly incorporated fragment. Transformed cells were grown at 30° C on plates containing ampicillin to confirm successful transformation. From these plates, colonies were selected and grown at 37° C on LB plates. Colonies from these plates were then grown on separate plates
containing ampicillin/LB, kanamycin/LB, and LB to confirm removal of the Kmr cassette and loss of pCP20.

D. Quantification of gene expression using β-galactosidase assays. Cells which simultaneously lost both ampicillin and kanamycin resistance were selected and used to perform β-galactosidase assays to quantify gene expression of our recombinant cells. Two overnight cultures of mutant cells and wild type MG1655 cells were grown to be used in the assay, one group containing IPTG and the other group with no IPTG. IPTG was controlled to demonstrate a loss of regulation by the lac operon repressor, lacI, resulting in constitutive expression of β–galactosidase.

Results

We observed dramatically lower levels of β–galactosidase expression in each of the mutants and loss of sensitivity to IPTG. Mutants containing the promoter sequence from plasmids pMP1-2 and pMP1-3 showed β–galactosidase activity of 5.87 and 7.50 Miller Units, respectively (Figure 1). Previous work on this project synthesized pMP1-1, which unsuccessfully altered β–galactosidase activity.

Discussion

Based upon this success, our project is being continued by repeating the above protocol to create a library of plasmids capable of altering gene expression in increasing amounts relative to plasmid pMP1-2 and pMP1-3. As our results demonstrate, we have been able to successfully downregulate the downstream gene of our promoters. However, we want to create a promoter capable of significantly increasing expression of any selected chromosomal gene. To do so, we are working with one mutant promoter sequence that significantly differs from our previously synthesized sequences based on the work of Jensen and Hammer. We plan to also introduce random sequences of DNA into our promoter region and screen for sequences which produce altered, constitutive expression of the genes downstream of the promoter.

Conclusion

Promoter swapping to control gene expression is a useful technique that can be used to understand cellular pathways. This technique can be an important tool in the field of metabolic engineering. We have created a successful protocol for insertion of two promotor sequences into the chromosome of Escherichia coli, causing constitutive expression of genes at altered levels. This project will continue by testing new sequences to create a twelve-plasmid library containing promoter sequences capable of altering gene expression in E. coli by varying amounts.

References

Datsenko, K. A., and B. L. Wanner. “One-step Inactivation of Chromosomal Genes in Escherichia Coli K-12 Using PCR Products.” Proceedings of the National Academy of Sciences 97.12 (2000): 6640-645. Web.

Jensen, P. R., and K. Hammer. “The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters.” Applied and Environmental Microbiology 64.1 (1998): 82-7. Print. McCleary, W.R. “Application of promoter swapping techniques to control expression of chromosomal genes.” Applied Microbiology Biotechnology 84 (2009): 641-648. Web.