Molecular biology has progressed at an amazing rate in the last two decades yielding a set of tools that allow us to manipulate DNA in a very controlled way. The aim of this section of the courses is to show a set of examples of the different tools that can be used to solve a wide variety of problems. Our claim is that, at least when dealing with E. coli, it is mostly about asking the right question rather than developing new techniques.

The particular goal of the project is to extract a gene from the E. coli genome (lacZ) and insert it into a plasmid that will control that gene under a tetracycline inducible promoter (pZE21, Lutz and Bujard, 1997). In order to accomplish this the students have to become acquainted with restricition enzymes, gel electrophoresis, PCR, ligation, transformations, sequencing and measuring gene expression at the single cell level using GFP.

Besides the obvious objective of manipulating DNA we had an extra question in mind. As an introduction to measuring gene expression we raised the question: "does the relative change in gene expression depend on the reporter gene that is used?". The relevance of this question is obvious if one is trying to be quantitative... the message should not depend on the messenger! This particular question is one of the advanced projects that some students take place in towards the end of the bootcamp.

Careful planning and understanding of the involved processes and their results is needed. We used Invitrogen's VectorNTI software in an intensive way, which allowed us to graphically understand what would happen to the DNA molecules in each step of this series of experiments. During these types of tutorials all students get to use the software on their computers and plan the next steps for the particular DNA molecule they will do cloning on.

Vector NTI allows to view commented DNA, and its features making it easy to predict restriction fragments, design PCR or sequencing primers and do BLAST searches of particular features of the DNA sequence we might be interested in.

General Concept

We used the pZ system developed by Lutz and Bujard as our vector. Its main advantage is that it has unique restriction sites around each one of its important features (coding region, antibiotic resistance, origin of replication, etc.), making it a highly modular plasmid system.

We worked with pZE21-GFP which expresses GFP under the control of a tetracycline inducible promoter (PLtetO-1). The plasmid also has resistance against the antibiotic kanamycin and its origin of replication is ColE1 (50-70 copies per cell).

Restriction Digest and Gel Electrophoresis

The original vector was digested with the restriction enzymes KpnI and HindIII and the samples were run on an agarose gel. This is a good chance for the students to perform a gel calibration using a DNA ladder.

Polymerase Chain Reaction (PCR)

Primers were designed to amplify one of the fluorescent genes from other plasmids and to add the correct restriction sites on either side. In order to illustrate the amplification process we measured the quantity of PCR product as a function of time using two methods. First, the hard way, by stopping the PCR reaction each cycle and extracting small aliquotes which can then be run on a gel; second, the easy way, by doing a qPCR assay which gives a fluorescent signal proportional to the amount of DNA in the reaction at each time point.

Ligation and Transformation

The vector and the insert were ligated together using T4 Ligase. The product was transformed into DH5a cells, which lack the tetracycline repressor (TetR). The absence of this repressor means that, in the case of a successful ligation, the cells will express lacZ at a high level. It was then easy to screen for these colonies using X-Gal, which turns blue upon cleavage by LacZ. In the picture the difference between the white and blue colonies can be seen.

Colony isolation and plasmid sequencing

Finally, the students were shown how to analyze sequencing results. Using VectorNTI and sequences obtained in the Caltech Sequencing Facility we learned how to interpret chromatography data and how to create contigs.

This confirmed the success of our cloning and we end up with a construct that will allow us to use LacZ as a reporter for the level of gene expression as opposed to using GFP, which is the gene that we had in the original construct.

Measuring Gene Expression at the Single Cell Level

The students measured the fluorescense of E. coli carrying pZE21-GFP (GFP induced by tetracycline) as a function of the concentration of tetracycline. The approach was to take phase contrast and fluorescence images of the same field of view. The students wrote custom Matlab which allowed them to recognize the cells in the phase contrast image and then integrate the GFP fluorescence over the area of each cell.

With this tool we can now obtain single cell statistics such as the average fluorescence per cell as a function of the concentration of inducer (ATC).

References:

Lutz R and Bujard H, Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements, Nucleic Acids Research, 1997; 25(6):1203-10.