Team:University of Washington/Project

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Bacterial Conjugation

The bacteria will then build the conjugation machinery, in response to the AHL produced from the yeast. This is being worked on in two separate ways on two different genes.

Two major genes in regulating IncP conjugation are KorA, a global regulation located on the RP4 plasmid, and TrbA, a gene that produces a transcriptional factor that controls the transcription of the TrbB operon, which contains the essential genes for building the conjugation pili and other parts of the conjugation tube.

Illustration of Tra1 Conjugation Machinery

Schröder, G., S. Krause, E. L. Zechner, B. Traxler, H. J. Yeo, R. Lurz, G. Waksman, and E. Lanka. 2002. TraG-like proteins of DNA transfer systems and of the Helicobacter pylori type IV secretion system: inner membrane gate for exported substrates? J. Bacteriol. 184:2767-2779. [http://www.ncbi.nlm.nih.gov/pubmed/11976307].

Yeast Shuttle Vector

One of the sub-projects involved in our controlled trans-kingdom conjugation project is the construction of a yeast shuttle vector. This plasmid will be stored and maintained in E. coli, and transferred to yeast via conjugation. Susbsequently, the plasmid will also be stored and maintained in yeast cells. Therefore, this yeast shuttle plasmid will have a bacterial origin of replication, a stable yeast origin of replication, a conjugative origin of transfer for RP4 mobilization, two genes which confer ampicilin and tetracycline resistance, a gene which imparts the ability to synthesize leucine, and finally the lac operon, which bestows the ability to digest lactose. A preliminary version of this plasmid, termed pAC88, has been created during prior research [7]. This pAC88 plasmid is a modified version of standard yeast plasmid YEp13; the origin of transfer site, OriT, from mobile plasmid RP4 was inserted into YEp13.

Functional pAC88 plasmids have been obtained from researchers at Leicester, which is a standard yeast cloning vector, YEp13, with an origin of transfer for RP4. The insertion of the RP4 OriT interrupts the gene for tetracycline resistance on YEp13, which is verifiable by transformed cells' sensitivity to tetracycline. In order to determine the efficacy of conjugation, S. cerevisiae was grown with E. coli in leucine-deficient media. Because pAC88 contains a gene which confers the ability to synthesize leucine, a strain of yeast which is normally auxotrophic for this amino acid was used. Successful conjugations resulted in yeast colonies that contained leucine.

There still remains the task of inserting the adaptive gene into the shuttle plasmid. Kluyveromyces lactis, a species of milk-yeast, posesses a region of chromosomal DNA that encodes for lactose digestion machinery. LAC4 and LAC12, two genes that encode for yeast galactosidase and lactose permease, respectively, are located near each other on the chromosome, and are divergently transcribed. Primers will be designed which are complementary to the transcriptional endpoints of LAC4 and LAC12, with hanging ends that correspond to the restriction enzyme HinDIII, an exploitable restriction site present in pAC88. A restriction digest on pAC88 and ligation with the LAC4-LAC12 cassette will insert the lactose digestion machinery into the yeast shuttle plasmid. By growing transformed yeast on a medium containing the molecular species X-gal, it will be determined whether the cassette has been successfully inserted if the yeast colonies appear blue.

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 <blockquote>'''Illustration of Tra1 Conjugation Machinery'''<br\></blockquote>
 <blockquote><p>Schröder, G., S. Krause, E. L. Zechner, B. Traxler, H. J. Yeo, R. Lurz, G. Waksman, and E. Lanka. 2002. TraG-like proteins of DNA transfer systems and of the Helicobacter pylori type IV secretion system: inner membrane gate for exported substrates? J. Bacteriol. 184:2767-2779. [http://www.ncbi.nlm.nih.gov/pubmed/11976307].</p></blockquote>
+== Yeast Shuttle Vector ==
+One of the sub-projects involved in our controlled trans-kingdom conjugation project is the construction of a yeast shuttle vector. This plasmid will be stored and maintained in E. coli, and transferred to yeast via conjugation. Susbsequently, the plasmid will also be stored and maintained in yeast cells. Therefore, this yeast shuttle plasmid will have a bacterial origin of replication, a stable yeast origin of replication, a conjugative origin of transfer for RP4 mobilization, two genes which confer ampicilin and tetracycline resistance, a gene which imparts the ability to synthesize leucine, and finally the lac operon, which bestows the ability to digest lactose. A preliminary version of this plasmid, termed pAC88, has been created during prior research [7]. This pAC88 plasmid is a modified version of standard yeast plasmid YEp13; the origin of transfer site, OriT, from mobile plasmid RP4 was inserted into YEp13.
+Functional pAC88 plasmids have been obtained from researchers at Leicester, which is a standard yeast cloning vector, YEp13, with an origin of transfer for RP4. The insertion of the RP4 OriT interrupts the gene for tetracycline resistance on YEp13, which is verifiable by transformed cells' sensitivity to tetracycline. In order to determine the efficacy of conjugation, S. cerevisiae was grown with E. coli in leucine-deficient media. Because pAC88 contains a gene which confers the ability to synthesize leucine, a strain of yeast which is normally auxotrophic for this amino acid was used. Successful conjugations resulted in yeast colonies that contained leucine.
+There still remains the task of inserting the adaptive gene into the shuttle plasmid. Kluyveromyces lactis, a species of milk-yeast, posesses a region of chromosomal DNA that encodes for lactose digestion machinery. LAC4 and LAC12, two genes that encode for yeast galactosidase and lactose permease, respectively, are located near each other on the chromosome, and are divergently transcribed. Primers will be designed which are complementary to the transcriptional endpoints of LAC4 and LAC12, with hanging ends that correspond to the restriction enzyme HinDIII, an exploitable restriction site present in pAC88. A restriction digest on pAC88 and ligation with the LAC4-LAC12 cassette will insert the lactose digestion machinery into the yeast shuttle plasmid. By growing transformed yeast on a medium containing the molecular species X-gal, it will be determined whether the cassette has been successfully inserted if the yeast colonies appear blue.
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