Caltech/Project/Q

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< Caltech | Project(Difference between revisions)
 
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==Role in Lambda Life Cycle==
==Role in Lambda Life Cycle==
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[[Image:Bacteriophage_lambda_genome.jpg|right|thumb|Schematic representation of the lambda genome. Q alters RNA polymerase and allows transcription of genes coding for heads and tails production.]]
Since Q is located downstream of the terminator tR, its transcription requires the presence of N.  During early stages of lambda's life cycle, as well as in the lysogenic pathway, the translation of Q is downregulated by an antisense RNA. The promoter pAQ transcribes Q in reverse, producing a Q-antisense RNA that binds to the Q messenger RNA and prevents ribosomal attachment.  Furthermore, a relatively high concentration of anti-Q is required to effectively cause antitermination, giving the phage time to begin a lysogenic life cycle if conditions are appropriate.
Since Q is located downstream of the terminator tR, its transcription requires the presence of N.  During early stages of lambda's life cycle, as well as in the lysogenic pathway, the translation of Q is downregulated by an antisense RNA. The promoter pAQ transcribes Q in reverse, producing a Q-antisense RNA that binds to the Q messenger RNA and prevents ribosomal attachment.  Furthermore, a relatively high concentration of anti-Q is required to effectively cause antitermination, giving the phage time to begin a lysogenic life cycle if conditions are appropriate.
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.
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[[Image:Bacteriophage_lambda_genome.jpg|center|frame|Schematic representation of the genome of the bacteriophage lambda.  Q alters RNA polymerase and allows transcription of genes coding for heads and tails production.]]
 
==Original Goals==
==Original Goals==
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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.
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The goal of this subproject is to determine the approximate concentration of Q necessary to allow infection. The Q gene cloned into a tetracycline-inducible expression system as described [[Caltech/Project/Constructs|here]]. ''E. coli'' cells containing the Q construct were titered with ''Q'' amber mutants, and the production of Q in the host cell was controlled by varying the concentration of the inducer aTc.
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==Status and Future Plans==
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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.
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[[Image:Q_results.jpg|center|Titering results for Q construct without cis repressor]]
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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.
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==Results==
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Titration trials with Q amber mutants have shown that expression of Q under tetR repression is leaky. In the absence of aTc, we still observed lysis using Q amber mutant phage. Replacing the spacer with various [[Caltech/Project/Riboregulator|cis-repressor]] sequences has shut down the leaky expression. Even in the presence of saturating aTc, no lysis was observed using the cis3 and cis4 repressed Q constructs.
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[[Image:Q_Construct.jpg|The Q construct used to determine concentration of Q necessary for lytic pathway induction.]]
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[[Image:Q_results.jpg|center|frame|Titering results using the Q amber mutant on D1210 ''E. coli'' containing the Q construct]]
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[[Image:cisQ_results.jpg|center|frame|Titering results using the Q amber mutant on D1210 ''E. coli'' containing the cis-repressed Q constructs]]
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We are currently working to introduce the conjugate trans activators to the cis3-repressed Q construct.
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Latest revision as of 00:46, 27 October 2007

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iGEM 2007

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Q-Antiterminator

Background Information

Q is a 207 amino acid protein with a mostly linear structure. It binds to lambda genomic DNA at Qut. The RNA polymerase is modified as it initializes transcription at pR', slightly downstream of Q. This modification causes the RNA polymerase to transcribe genes beyond the terminator tR'.

Role in Lambda Life Cycle

Schematic representation of the lambda genome. Q alters RNA polymerase and allows transcription of genes coding for heads and tails production.

Since Q is located downstream of the terminator tR, its transcription requires the presence of N. During early stages of lambda's life cycle, as well as in the lysogenic pathway, the translation of Q is downregulated by an antisense RNA. The promoter pAQ transcribes Q in reverse, producing a Q-antisense RNA that binds to the Q messenger RNA and prevents ribosomal attachment. Furthermore, a relatively high concentration of anti-Q is required to effectively cause antitermination, giving the phage time to begin a lysogenic life cycle if conditions are appropriate.

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.

Original Goals

The goal of this subproject is to determine the approximate concentration of Q necessary to allow infection. The Q gene cloned into a tetracycline-inducible expression system as described here. E. coli cells containing the Q construct were titered with Q amber mutants, and the production of Q in the host cell was controlled by varying the concentration of the inducer aTc.

Results

Titration trials with Q amber mutants have shown that expression of Q under tetR repression is leaky. In the absence of aTc, we still observed lysis using Q amber mutant phage. Replacing the spacer with various cis-repressor sequences has shut down the leaky expression. Even in the presence of saturating aTc, no lysis was observed using the cis3 and cis4 repressed Q constructs.

Titering results using the Q amber mutant on D1210 E. coli containing the Q construct
Titering results using the Q amber mutant on D1210 E. coli containing the cis-repressed Q constructs

We are currently working to introduce the conjugate trans activators to the cis3-repressed Q construct.

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