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Plasmid Selection Notes

submitted by Christian Ling

When selecting a suitable plasmid for our experiment our team decided to work backwards, in a sense, by thinking about our end product first in order to determine what qualities it is that our plasmid needs. Here is a short list of elements that we deemed as desirable in the plasmid to be selected:

1. The plasmid CAN be properly replicated in S. oneidensis and will persist in future generations of transformed bacteria.
2. The plasmid contains oriT and oriV for conjugal transfer of the plasmid from E. coli to S. oneidensis and vegetative replication following transfer, respectively.
3. A minimal number of other major genes in order to avoid confounding of results due to extra or unexpected gene expression, or combinations of gene expression.
4. A robust number of cleavage sites which will allow for the transposing of two genes into the plasmid.

With the above criteria in mind, the team agreed upon using plasmid pJQ200, sold by bioresource company ATCC [http://www.atcc.org/catalog/numSearch/numResults.cfm?atccNum=77482], for the following reasons:

1. Due to the presence of origin of replication p15A, plasmid pJQ200 will readily replicate in both E. coli and S. oneidensis. This facilitates the use of selection methods to "breed" high performing strains of S. oneidensis.
2. pJQ200 contains oriT and oriV, allowing for conjugation.
3. The only major genes listed by the ATCC for pJQ200 are sacB, gmR, and traJ.
   1. traJ refers to transfer genes which are necessary for conjugation to occur. However, the E.coli strain S17-1, which we will be using in conjugation, is already capable of conjugation. This makes the traJ gene redundant in the E.coli. Otherwise, in the transformed S. oneidensis traJ will not interfere with current production as it is only linked to conjugation.
   2. The gmR gene is actually undesirable as the strain of S. oneidensis we are using, MR-1, already contains gentamicin resistance. gmR in the plasmid would inhibit our ability to select for transformed S. oneidensis using double antibiotic control plates. Therefore, we will be capitalizing on the presence of restriction sites Tth111I and Eco31I (though during digestion we will be using BsaI, which is an isoschizomer of Eco31I) within the gmR gene to cut out the gene and insert a kanR gene.
   3. The sacB site confers lethal sucrose sensitivity in gram-negative bacteria/non-enterobacteria. S. oneidensis falls into this category. Since we intend on transposing the global transcription regulators into the sacB gene site (via restriction sites HindIII and EcoRI), we can select for integration of the transcription regulators into the sacB gene by incorporating sucrose into our agar plates.
4. There are an excellent selection of restriction sites on pJQ200, as evinced by all the restriction sites mentioned above.

Here is a map of the plasmid with restriction sites and major genes marked.

http://www.atcc.org/common/images/vectors/gifs/77482.gif

New England Biolabs (NEB) Cutter Notes

NEB provides a NEBCutter website that allows users to freely check nucleotide sequences for restriction enzyme sites. Features include a graphical representation of sequences/plasmids with all applicable cut sites, the ability to enter in custom recognition sites for restriction enzymes, and an option to use sequences in the NCBI GenBank. Below are some brief instructions on using the NEBCutter for checking specific, user-defined restriction sites.

Procedure to use NEBCutter for checking specific restriction enzymes against a sequence submitted by Christian Ling

1. Bring up the NEBCutter page at <http://tools.neb.com/NEBcutter2/index.php>

2. Enter in your nucleotide sequence by either uploading a 'local sequence' file from your computer, inputting a
valid GenBank number, or manually entering/pasting in the nucleotide sequence into the large text box given.

3. To check the sequence against specific restriction enzymes, first click the radio button for "Only Defined
Oligonucleotide Sequences" when choosing which "Enzymes to Use".

4. Then click on the [define oligos] link. A pop-up window should appear in which multiple enzymes can be named in
the left column,and have their recognition sequence defined in the right column. Fill out all the necessary
information for the enzymes you will be using and click the 'OK' button.

5. Select whether the sequence is linear or circular and specify the minimum O.R.F length to display.

6. Name the sequence using the "Name of Sequence" text box in the middle of the page.

7. Hit "Submit" and wait for your results.

Making Electrophoresis Gel

1. Take 60ml TAE 1X and add 0.6g agarose. 2. Microwave for about 45 seconds. 3. Let it cool until its cool enough so you can touch it for an extended period of time. 4. Add 4ul of Etbr and pour it on the gel-plate.

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