IGEM:Caltech/2007/Project/Q: Difference between revisions
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=<center>Q-Antiterminator</center>= | |||
==Background Information== | ==Background Information== | ||
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Once Q has modified the RNA polymerase, the polymerase begins to transcribe genes beyond tR'. These genes are crucial to the production of new phage components, such as tails and heads (protein capsules). Once these genes are expressed, the phage is committed to the lytic pathway. | Once Q has modified the RNA polymerase, the polymerase begins to transcribe genes beyond tR'. These genes are crucial to the production of new phage components, such as tails and heads (protein capsules). Once these genes are expressed, the phage is committed to the lytic pathway. | ||
[[Image:Bacteriophage_lambda_genome.png|thumb|center|Schematic representation of the genome of the bacteriophage lambda. Q alters RNA polymerase and allows transcription of genes coding for heads and tails production.]] | |||
[[Image:Q_changes.jpg|center|changes brought on by the Q protein]] | |||
==Original Goals== | ==Original Goals== | ||
The goal of this subproject is to determine the approximate concentration of Q necessary to effectively switch from a lysogenic to a lytic pathway. ''Q'' amber mutants, which are deficient in the production of Q, will be used, and the production of Q in the host cell will be controlled by varying the concentration of inducer ATC. The fraction of colonies that lyse will be measured, and a graph of lysis vs. Q production will be obtained. | The goal of this subproject is to determine the approximate concentration of Q necessary to effectively switch from a lysogenic to a lytic pathway. ''Q'' amber mutants, which are deficient in the production of Q, will be used, and the production of Q in the host cell will be controlled by varying the concentration of inducer ATC. The fraction of colonies that lyse will be measured, and a graph of lysis vs. Q production will be obtained. | ||
== | ==Status and Future Plans== | ||
The Q constructs, consisting of promoter pTet, spacer, Q gene, double terminator, constitutive promoter, tetR gene, and double terminator have been made and sequence verified. There are two version of the construct, one with a medium-strength constitutive promoter driving tetR, and one with a strong constitutive promoter. Titration trials with Q amber mutants have shown that expression of Q under tetR repression using the medium-strength promoter is leaky but inducible with anhydrotetracycline (aTc). Replacing the spacer with various cis-repressor sequences has shut down the leaky repression. | |||
[[Image:Q_results.jpg|center|Titering results for Q construct without cis repressor]] | |||
Future work on Q includes titering the construct featuring the strong constitutive promoter, introducing the conjugate trans activators to Q construct variants containing cis repressors, and quantitatively measuring the effects of aTc concentration on cell lysis rates. | |||
[[Image:Q_Construct.jpg|The Q construct used to determine concentration of Q necessary for lytic pathway induction.]] | |||
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Latest revision as of 00:18, 26 October 2007
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