introduction/background Bio lab paper

1. ORF fusions between GFP and a Target Gene

2. pPEM109

3. LEPA/EF4; elongation factor 4 (EF-4) refer to the gene as lepA

4. Tn5 Vitro transposition reaction: http://www.jbc.org/content/273/13/7367.full

5. TRANSFORMATION OF TRANSPOSITION MIXTURE INTO E. coli.

Read those overviews to have an idea about the topics!

III. Creating ORF fusions between GFP and a Target Gene

This experimental module has the aim of creating novel Green Fluorescent Protein (GFP) gene fusions using a simple one step insertion method. The method employs in vitro transposition using Tn5 transposase to obtain randomly generated in frame fusions between a target gene and the GFP gene. The in vitro transposition reaction requires two pieces of DNA; a donor transposon and a target. The transposition step will generate a library of products containing the donor transposon piece of DNA randomly inserted into the target DNA. The donor transposon DNA will be synthesized in the lab using the Polymerase Chain Reaction. The design of this synthetic DNA includes a GFP open reading frame (ORF) that has no promoter, no start codon, and no stop codon so that production of GFP protein will require in frame insertion into an ORF in the target DNA. Therefore, even

it is a single-celled organism, making it cheap and easy to work with in the lab
it is able to reproduce and grow very rapidly, doubling every 20 minutes
it has a relatively small genome size, allowing many strains to be fully sequenced it is genetically tractable, allowing genes to be introduced, removed, or engineered

will be employed as a model organism in experiments that will introduce core methods and concepts used in research in molecular biology, genetics, and biochemistry. Topics will include: the use of microbial methods to grow cultures of bacteria, isolation of DNA and protein from bacteria, DNA and protein gel electrophoresis, western blotting, the use of plasmids for cloning and gene expression, the Polymerase Chain Reaction (PCR), gene structure and function analysis, transcription, translation, protein structure, and creation of altered genes via recombinant DNA procedures.

Creating ORF fusions between GFP & LepA Gene

though Tn5 catalyzed in vitro transposition is random, in frame ORF fusions can be recovered simply by transforming the transposition reaction library into cells. Resulting colonies are screened for GFP fluorescence directly by exposure to ultraviolet light.

These experiments will employ the E. coli gene lepA as the target DNA, which encodes the translation elongation protein factor EF-4. The lepA gene used in these experiments is present on a plasmid, pPEM109, that was prepared during prior research.

Tn5 Transposition

Transposition of donor DNA catalyzed by the protein Tn5 transposase requires that the donor DNA molecule contain key elements from the Tn5 transposon. Essential features of the Tn5 transposon include specific 19 bp inverted, repeated, DNA sequences flanking the 5’ and 3’ ends of the donor DNA (FIGURE 1A).

1 GFP Donor DNA

A

1. Individual molecules of transposase (blue spheres) bind to specific sites (black) at the ends of the transposon DNA.

2. Looping of the transposon DNA results in formation of a complex that brings the two ends of the transposable element close together.

3. Tn5 transposase/DNA complex can move about freely until it encounters and binds to the "target" DNA (red).

pPEM109 target DNA

4. Transposase catalyzes insertion of the transposon DNA into the target DNA, completing the transposition.

2. Transposase binding

3. Target capture

4. Strand transfer

FIGURE 1. A) Mechanism of transposition by Tn5 transposase. Source: Adapted from University of Wisconsin-Madison. B) Schematic of transposition reaction to randomly insert GFP ORF into target DNA plasmid (red circle). The 19 bp inverted repeats (black rectangles) sequences are found at each end of the GFP gene sequence. The GFP gene is represented by the green bar.

The target DNA can be any double stranded piece of DNA. The target DNA employed in this module is a plasmid containing the E. coli gene that encodes LepA/EF-4. The name of this plasmid is pPEM109. pPEM109 is represented in FIGURE 1B by the red double circle.

Overview: Initially the target plasmid (pPEM109) containing the lepA gene will be isolated and donor transposon DNA will be synthesized. After characterizing these pieces of DNA, they will be combined with Tn5 transposase. This reaction will generate an insertion library as illustrated in FIGURE 1 above. This library will be transformed into E. coli in order to isolate insertion-positive candidates by screening for fluorescent colonies growing on plates. The plasmid derived from fluorescent colonies will be purified and compared to the original target DNA. The characterization will include determining the relative sizes of the plasmid DNA, and the EF-4 protein produced, DNA sequence analysis, and western blotting to prove that the insertion-positive candidate contains GFP sequence. DNA sequence information will be used, in conjunction with information about the three- dimensional protein structure to determine where the GFP sequence is inserted within the structure of EF-4. Functional genetic and biochemical assays will be performed to assess whether the newly created GFP fusion proteins are active.

PLASMID PURIFICATION, PURIFICATION OF TARGET DNA. LepA/EF-4 GFP tagging project introduction, plasmid purification.

Purpose: To amplilfy and purify plasmid DNA containing lepA gene as a GFP target for an in vitro Tn 5 transposase-catalyzed transposition reaction

Background: The major goal of this research project is to use GFP as a biological marker to study Lep1/EF-4 function. We will use Tn5 transposase to introduce the GFP donor DNA into the lepA target DNA, which is stored inside the plasmid vector pPEM109. To obtain sufficient quantities of target plasmid DNA containing the lepA gene for transposition, we will allow bacteria containing the plasmid to divide many times in liquid culture and then break open cells, purifying plasmid DNA away from other cellular components. Click the following link to learn how and why plasmid purification protocol works: http://vimeo.com/37329445

Purpose: To introduce a mixed library of GFP insertion plasmids into bacteria to measure

GFP fluorescence in vivo

Background: (Adapted from AddGene) Transformation is the process by which foreign DNA is introduced into a cell (FIGURE 6). Transformation of bacteria with plasmids is important not only for studies in bacteria but also because bacteria are used as the means for both storing and replicating plasmids. Because of this, nearly all plasmids, even those designed for use in mammalian cells, carry both a bacterial origin of replication and an antibiotic resistance gene for use as a selectable marker in bacteria.

Scientists have made many genetic modifications to create bacterial strains that can be more easily transformed and maintain the plasmid without rearrangement of the plasmid DNA. Additionally, treatments have been discovered that increase the transformation efficiency of the bacteria and make them more susceptible to either chemical or electrical based transformation, generating what are commonly referred to as ‘competent cells’.

Click the following link to learn how and why the bacteria transformation protocol works:

http://www.dnalc.org/view/15918-Transformation.html