Team:UC Davis/Project

From 2011.igem.org

(Difference between revisions)
 
(37 intermediate revisions not shown)
Line 18: Line 18:
<h1>Overview</h1>
<h1>Overview</h1>
<div class="floatbox3">
<div class="floatbox3">
-
We set out to develop a rugged, easy-to-use mutagenic PCR protocol for the rapid production of mutant libraries of any <a href="http://biobricks.org/" target:"_blank">BioBrick</a> part using standard primers, and to prototype this protocol by creating mutant libraries of the LacI, TetR and λ cI repressible promoters.
+
We set out to develop a quick, easy process for the expansion of basic parts into a part families. Our method employs a suped-up <a href="https://2011.igem.org/Team:UC_Davis/Protocols#ER-PCR">mutagenic PCR protocol</a> that uses standard VF2 and VR primers and materials most iGEM teams already have on hand. We chose to prototype this process by creating a part family from the LacI promoter R0010, and to mutate GFP to visually assess our ability to create functional protein mutants.<br><br>
-
</div>
+
-
<div class="floatbox2">
+
As of November 2011, we have a functioning part family generation process and seven <a href="https://2011.igem.org/Team:UC_Davis/Data_LacI">well-characterized</a> LacI promoter mutants and eight GFP mutants (two of which have been lovingly named Orange-Mutated Green Fluorescent Protein or [OMGfp] 1 and 2) which await further characterization.<br><br>
-
<h2 id="whyMutants">Why Make Mutant Libraries?</h2>
+
-
When designing novel genetic circuits, synthetic biologists are limited to using parts that are already available or that they can manufacture themselves. The creation of new parts can be incredibly time-consuming, since they must be extracted from existing natural genes and converted to a DNA part standard (like our <a href="http://biobricks.org/" target:"_blank">BioBricks</a>) or modified from existing parts.<br><br>
+
-
The <a href="http://www.partsregistry.org" target="_blank">parts registry</a> contains many useful parts, but in cases where fine control over the function of a part is required for a genetic circuit to function properly, the existing selection may not offer enough variation in activity, chemical response, or other characteristics.<br><br>
+
We have also begun the process of generating part families from the TetR (R0040) and Lambda c1 (R0051) promoters, which are currently being selected for screening.
 +
</div>
-
<div class="floatbox2">
+
<div class="floatboxleft">
-
<h2 id="libraries">Generating Mutant Libraries with Error-Prone PCR</h2>
+
<h2>Project Selection</h2>
-
The process by which we produce our mutant libraries is both fast and simple. We select a BioBrick part and prepare an error-prone PCR reaction using a small amount of template, standard VF2 and VR primers, the <a href=https://2011.igem.org/Team:UC_Davis/Protocols#ER-PCR>error-prone PCR mix</a> listed on our <a href="https://2011.igem.org/Team:UC_Davis/Protocols">protocols</a> page and Taq polymerase enzyme. The products of this reaction may be diluted and run through another round of PCR to introduce more mutations.<br><br>  
+
Why would we want to make mutants? What's so special about repressible promoters? Read about how we came to decide on this project idea <a href="https://2011.igem.org/Team:UC_Davis/Project_Selection">here.</a><br><br>
-
Mutant parts are then ligated directly into a screening construct like those listed below to help assess part activity. For example, mutant promoters can be placed in front of GFP to estimate their strength by relative fluorescence, whereas repressors can be paired with their corresponding promoter in front of a reporter. Transformation of these ligation products into competent DH5-α cells yields plates with many colonies, many containing mutants of the selected BioBrick part.<br><br>
+
</div>
-
Colonies with the desired level of reporter expression are transferred to replica plates, grown up in liquid culture, and assayed in a 96-well plate reader. Consecutive screens are performed to narrow down the number of potential mutants so that they may be run in triplicate in our plate reader, using Octave scripts to select mutants with the desired range of activity.<br><br>
+
-
Once a final group of mutants has been picked, they are carefully characterized for their activity. You can read more about the characterization process for our promoter mutants <a>here</a>.
+
-
<!--To test our protocol's ability to produce functional protein mutants, we ran E0240 (GFP) through one round of our error-prone PCR procedure and performed visual inspections of the resulting colonies for their variation from wild-type GFP.<br><br>--!>
+
<div class="floatboxright">
 +
<h2>Make a Part Family</h2>
 +
Want to make your own mutant library? Curious as to how we assayed our promoter mutants, or how we selected variants that had well-spaced activity levels? Read about our library generation process <a href="https://2011.igem.org/Team:UC_Davis/PartFamilies">here</a>. <br>
 +
</div>
-
</div>
+
<div class="floatboxleft">
 +
<h2>Promoter Mutants</h2>
 +
We chose to mutate repressible promoters because they are useful in designing complex circuits and are relatively easy to screen for changes in activity level. You can view detailed information on our <a href="https://2011.igem.org/Team:UC_Davis/PromoterFamilies#LacI">LacI</a>, <a href="https://2011.igem.org/Team:UC_Davis/PromoterFamilies#TetR">TetR</a> and <a href="https://2011.igem.org/Team:UC_Davis/PromoterFamilies#c1">λ cI</a> part families on their respective pages.<br><br>
</div>
</div>
-
<div class="floatbox">
+
<div class="floatboxright">
-
<h1 id ="promotermut">Promoter and Repressor Mutants</h1>
+
<h2>KO3D Plotting Library</h2>
-
To show that our protocol can also be used to diversify the application of promoters and other regulatory gene sequences, we performed our mutagenic PCR process on parts R0010, R0040 and R0051 (the LacI, TetR and c1 Lambda promoters) using multiple successive rounds of error-prone PCR. We screened these mutants for promoter activity compared to wild type, and characterized their response to changes in repressor concentration. We also characterized the response of wild-type and mutant LacI promoter / wild-type repressor pairs to IPTG at various repressor concentration levels.
+
When researching ways to present <a href="https://2011.igem.org/Team:UC_Davis/Data_LacI">LacI characterization data</a> clearly on this website, we realized there were no simple, cross-platform javascript libraries for interactive 3D data plotting. To rectify this, we coded our own. Read more about it <a href="https://2011.igem.org/Team:UC_Davis/KO3D">here</a>.
-
<div class="floatbox2">
+
-
<h2 id="construct">General Mutant Screening Constructs</h2>
+
-
The specific order in which the parts are depicted below allows the user to swap in any promoter/repressor or promoter/activator pair using our regulatory characterization plasmid, K611018.<br><br>
+
-
<center><img src="https://static.igem.org/mediawiki/2011/2/21/UCD_Gen_pMut_construct.png"></center><br><br>
+
-
We designed this construct for characterizing promoter mutants. When pBAD is induced with arabinose, the repressor of choice is transcribed leading to decreased levels of the reporter, GFP.<br><br> 
+
-
<center><img src="https://static.igem.org/mediawiki/2011/1/12/UCD_Gen_rMut_construct.png"><br><br></center>
+
-
The mutant repressor characterization construct is nearly identical to its promoter-based counterpart. The user simply ligates the wild-type promoter to the 5' end and a mutant repressor or activator to the 3' end.
+
-
 
+
-
</div>
+
</div>
</div>
 +
<style>
<style>

Latest revision as of 20:15, 23 October 2011

Our Sponsors

Start a Family

Got a favorite BioBrick? Check our our process for expanding basic parts into part families.

Criteria

View our judging criteria for iGEM 2011 here.

Overview

We set out to develop a quick, easy process for the expansion of basic parts into a part families. Our method employs a suped-up mutagenic PCR protocol that uses standard VF2 and VR primers and materials most iGEM teams already have on hand. We chose to prototype this process by creating a part family from the LacI promoter R0010, and to mutate GFP to visually assess our ability to create functional protein mutants.

As of November 2011, we have a functioning part family generation process and seven well-characterized LacI promoter mutants and eight GFP mutants (two of which have been lovingly named Orange-Mutated Green Fluorescent Protein or [OMGfp] 1 and 2) which await further characterization.

We have also begun the process of generating part families from the TetR (R0040) and Lambda c1 (R0051) promoters, which are currently being selected for screening.

Project Selection

Why would we want to make mutants? What's so special about repressible promoters? Read about how we came to decide on this project idea here.

Make a Part Family

Want to make your own mutant library? Curious as to how we assayed our promoter mutants, or how we selected variants that had well-spaced activity levels? Read about our library generation process here.

Promoter Mutants

We chose to mutate repressible promoters because they are useful in designing complex circuits and are relatively easy to screen for changes in activity level. You can view detailed information on our LacI, TetR and λ cI part families on their respective pages.

KO3D Plotting Library

When researching ways to present LacI characterization data clearly on this website, we realized there were no simple, cross-platform javascript libraries for interactive 3D data plotting. To rectify this, we coded our own. Read more about it here.

Retrieved from "http://2011.igem.org/Team:UC_Davis/Project"