Difference between revisions of "Team:William and Mary/Notebook"

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<div class="title">
 
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Description
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Notebook
 
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<div class="container">
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<div class="title add-animation-stopped">
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<p class='h2WM' style='padding-left:70px; padding-top: 0px;'>
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Motivation
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<div class="col-md-7 col-md-offset-1" style='padding-top: 0px;'>
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<p class='large' style="color:#A9A9A9;">
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  Genetic circuits exist in great abundance in nature as complex metabolic pathways which interact
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  in various ways to perform vital cellular processes. Synthetic biologists aim to not only understand
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  naturally occurring circuit networks, but also to modify them or to conceptualize and build entirely
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  new circuits.
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</p>
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<div class="description">
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<p class='large' style="padding-left:70px;">
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The inherent versatility of synthetic genetic circuitry has lead to a vast array of diverse applications
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in countless fields. However, the field remains fundamentally limited by the magnitude and specificity of
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behavioral control over genetic circuits and circuit networks. These limitations can be boiled down to two
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essential problems: inherent constraints to behavior based on the nature of a circuit’s constituent genes,
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and the inefficiency of the “design-build-test” cycle which is relied upon for the construction of effective
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circuit models.
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</p>
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<p class='large' style="padding-left:70px;">
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The fundamental constraints of integral circuit components limit the ability to design and construct
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genetic circuits of arbitrary and highly specific behavior. When constructing a circuit with some intended
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behavior, design is limited by the available input-specific regulators to gene expression and their
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characteristic regulatory behavior. In order to achieve more precise behavioral control, the ability to
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tune expression levels of regulatory elements to some desired level is vital. This limitation highlights
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the need for genetic devices that can modify the behavior of arbitrary genetic circuits; implementing these
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devices would enable precise behavioral control invariant to the constraints of the constituent genes that
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make up the circuit in question [1].
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</p>
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<p class='large' style="padding-left:70px; padding-bottom:50px;">
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The other foundational limitation of genetic circuit construction addresses the inefficiency and
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unpredictability of the design and construction process itself. The progression from synthesizing parts
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into a circuit on a plasmid, to transformation and testing in vivo, is a lengthy and expensive process
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which furthermore is largely variable in terms of actual functionality of the final product [2].This often
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leads to a series of trial-and-error testing cycles whose products maintain a persistent level of uncertainty
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with regard to precise, predictable behavior. Although it is possible to achieve functional genetic circuits
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in this capacity, greater problems arise regarding the tunability of the product. The success of any genetic
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circuit relies on the ability to precisely tune a response to a range of input concentrations; it would therefore
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be desirable to obtain a reliable method for tuning circuit response, ideally without the need to rewire the
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internal workings of the circuit. This method would allow control over output expression to be implemented in
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a more rapid and predictable manner [3].
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</p>
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<div class="col-md-4">
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<div class="title add-animation-stopped">
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<p class='h2WM' style='padding-left:70px;'>
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The Project
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</p>
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<div class="col-md-7 col-md-offset-1" style='padding-top: 0px;'>
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<div class="description add-animation-stopped animation-1">
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<p class='large' style="color:#A9A9A9;">
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Our project aims to provide a modular collection of genetic parts which can specifically and predictably
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tune the behavior of an arbitrary genetic circuit. This collection, which we have dubbed the “Circuit
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Control Toolbox,” consists of a suite of parts which can be added to the end of a given genetic circuit;
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each part provides a specific and independently tunable response which allows direct control over the
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ultimate output behavior of the circuit.
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</p>
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</div>
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<div class="description">
 
<div class="description">
 
<p class='large' style="padding-left:70px;">
 
<p class='large' style="padding-left:70px;">
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The overall input/output behavior of any genetic circuit can be represented by a graph known as a
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Description of this page
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transfer function, which relates concentration of input molecule to output protein expression.
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Likewise, any modifications to the circuit affecting input/output behavior can be visualized by a
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transformation of the transfer function representing the circuit. The Circuit Control Toolbox consists
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of three distinct tools which prompt unique behavioral changes to the circuit’s output relative to its
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input, and therefore generate different transformations of the circuit’s original transfer function.
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</p>
 
</p>
 
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<!--WEEK-->
 
<!--WEEK-->
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<p class='large' style="padding-left:70px; padding-bottom:50px;">
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<p class='large' style="padding-left:70px; padding-bottom:30px;">
 
Week 1 (160529 – 160604)
 
Week 1 (160529 – 160604)
 
</p>
 
</p>
 
 
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<p class='large' style="padding-left:70px; padding-bottom: 30px;">
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<p class='large' style="padding-left:90px; padding-bottom: 10px;">
 
<b>✻</b> Resuspended parts from the kit.
 
<b>✻</b> Resuspended parts from the kit.
 
</p>
 
</p>
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<p class='large' style="padding-left:70px; padding-bottom: 30px;">
+
<p class='large' style="padding-left:90px; padding-bottom: 10px;">
 
<b>✻</b> Resuspended gBlocks of ordered sequences.
 
<b>✻</b> Resuspended gBlocks of ordered sequences.
 
</p>
 
</p>
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<p class='large' style="padding-left:70px; padding-bottom: 30px;">
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<p class='large' style="padding-left:90px; padding-bottom: 10px;">
 
<b>✻</b> Created a functional UNS Standard Backbone, containing the UNS 2 and 3 sequences within the Prefix and Suffix.
 
<b>✻</b> Created a functional UNS Standard Backbone, containing the UNS 2 and 3 sequences within the Prefix and Suffix.
 
</p>
 
</p>
−
<p class='large' style="padding-left:70px; padding-bottom: 30px;">
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<p class='large' style="padding-left:90px; padding-bottom: 10px;">
 
<b>✻</b> Cloned K2066001-K2066016 into UNS pSB1X3 Backbone.
 
<b>✻</b> Cloned K2066001-K2066016 into UNS pSB1X3 Backbone.
 
</p>
 
</p>
−
<p class='large' style="padding-left:70px; padding-bottom: 30px;">
+
<p class='large' style="padding-left:90px; padding-bottom: 10px;">
 
<b>✻</b> Performed Diagnostic PCRs to test primer design for Ribozyme / RiboJ Characterization subproject
 
<b>✻</b> Performed Diagnostic PCRs to test primer design for Ribozyme / RiboJ Characterization subproject
 
</p>
 
</p>
−
<p class='large' style="padding-left:70px; padding-bottom: 30px;">
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<p class='large' style="padding-left:90px; padding-bottom: 10px;">
 
<b>✻</b> Transformed interlab devices, created glycerol stocks. Could not get IMP #1, #3, and (-) control to transform.  
 
<b>✻</b> Transformed interlab devices, created glycerol stocks. Could not get IMP #1, #3, and (-) control to transform.  
 
</p>
 
</p>
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<!--WEEK-->
 
<!--WEEK-->
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<p class='large' style="padding-left:70px; padding-bottom:50px;">
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<p class='large' style="padding-left:70px; padding-bottom:30px;">
 
Week 2 (160605 – 160611)
 
Week 2 (160605 – 160611)
 
</p>
 
</p>
 
 
−
<p class='large' style="padding-left:70px; padding-bottom: 30px;">
+
<p class='large' style="padding-left:90px; padding-bottom: 10px;">
 
<b>✻</b> Constructed K2066024, K2066025, and K2066027 using Gibson Assembly (from K2066014, K2066015, and pTAC templates).  
 
<b>✻</b> Constructed K2066024, K2066025, and K2066027 using Gibson Assembly (from K2066014, K2066015, and pTAC templates).  
 
</p>
 
</p>
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<p class='large' style="padding-left:70px; padding-bottom: 30px;">
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<p class='large' style="padding-left:90px; padding-bottom: 10px;">
 
<b>✻</b> We received training on the FACS (Fluorescence Activated Cell Sorter) Machine.  
 
<b>✻</b> We received training on the FACS (Fluorescence Activated Cell Sorter) Machine.  
 
</p>
 
</p>
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<p class='large' style="padding-left:70px; padding-bottom: 30px;">
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<p class='large' style="padding-left:90px; padding-bottom: 10px;">
 
<b>✻</b> Realized the need for repressor protein / fluorophore fusion proteins (LacI-mCherry, TetR-GFP, i.e.)
 
<b>✻</b> Realized the need for repressor protein / fluorophore fusion proteins (LacI-mCherry, TetR-GFP, i.e.)
 
</p>
 
</p>
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<p class='large' style="padding-left:70px; padding-bottom: 30px;">
+
<p class='large' style="padding-left:90px; padding-bottom: 10px;">
 
<b>✻</b> Designed gBlocks of K2066028 and K2066029.  
 
<b>✻</b> Designed gBlocks of K2066028 and K2066029.  
 
</p>
 
</p>
−
<p class='large' style="padding-left:70px; padding-bottom: 30px;">
+
<p class='large' style="padding-left:90px; padding-bottom: 10px;">
 
<b>✻</b> Attempted to clone Addgene 240x TetO Sequence into UNS Standard Backbone.  
 
<b>✻</b> Attempted to clone Addgene 240x TetO Sequence into UNS Standard Backbone.  
 
</p>
 
</p>
−
<p class='large' style="padding-left:70px; padding-bottom: 30px;">
+
<p class='large' style="padding-left:90px; padding-bottom: 10px;">
 
<b>✻</b> Attempted ICA with K2066002, K2066003, K2066004 to create a TetO w/ 8bp spacer 9-mer
 
<b>✻</b> Attempted ICA with K2066002, K2066003, K2066004 to create a TetO w/ 8bp spacer 9-mer
 
</p>
 
</p>
−
<p class='large' style="padding-left:70px; padding-bottom: 30px;">
+
<p class='large' style="padding-left:90px; padding-bottom: 10px;">
 
<b>✻</b> Constructed K2066030 and K2066031 from K2066015.
 
<b>✻</b> Constructed K2066030 and K2066031 from K2066015.
 
</p>
 
</p>
−
<p class='large' style="padding-left:70px; padding-bottom: 30px;">
+
<p class='large' style="padding-left:90px; padding-bottom: 10px;">
 
<b>✻</b> Troubleshot ICA, attempted 6- and 12-mers of TetO w/ 8bp spacer.  
 
<b>✻</b> Troubleshot ICA, attempted 6- and 12-mers of TetO w/ 8bp spacer.  
 
</p>
 
</p>
−
<p class='large' style="padding-left:70px; padding-bottom: 30px;">
+
<p class='large' style="padding-left:90px; padding-bottom: 10px;">
 
<b>✻</b> Attempted IPTG Induction of K2066014 + K2066016 cotransformations.
 
<b>✻</b> Attempted IPTG Induction of K2066014 + K2066016 cotransformations.
 
</p>
 
</p>
 
</p>
 
</p>
−
<p class='large' style="padding-left:70px; padding-bottom: 30px;">
+
<p class='large' style="padding-left:90px; padding-bottom: 10px;">
 
<b>✻</b> Transformed remaining interlab devices, created glycerol stocks.
 
<b>✻</b> Transformed remaining interlab devices, created glycerol stocks.
 
</p>
 
</p>

Revision as of 22:04, 19 October 2016


...

Notebook

Description of this page

Week 1 (160529 – 160604)

✻ Resuspended parts from the kit.

✻ Resuspended gBlocks of ordered sequences.

✻ Created a functional UNS Standard Backbone, containing the UNS 2 and 3 sequences within the Prefix and Suffix.

✻ Cloned K2066001-K2066016 into UNS pSB1X3 Backbone.

✻ Performed Diagnostic PCRs to test primer design for Ribozyme / RiboJ Characterization subproject

✻ Transformed interlab devices, created glycerol stocks. Could not get IMP #1, #3, and (-) control to transform.

Week 2 (160605 – 160611)

✻ Constructed K2066024, K2066025, and K2066027 using Gibson Assembly (from K2066014, K2066015, and pTAC templates).

✻ We received training on the FACS (Fluorescence Activated Cell Sorter) Machine.

✻ Realized the need for repressor protein / fluorophore fusion proteins (LacI-mCherry, TetR-GFP, i.e.)

✻ Designed gBlocks of K2066028 and K2066029.

✻ Attempted to clone Addgene 240x TetO Sequence into UNS Standard Backbone.

✻ Attempted ICA with K2066002, K2066003, K2066004 to create a TetO w/ 8bp spacer 9-mer

✻ Constructed K2066030 and K2066031 from K2066015.

✻ Troubleshot ICA, attempted 6- and 12-mers of TetO w/ 8bp spacer.

✻ Attempted IPTG Induction of K2066014 + K2066016 cotransformations.

✻ Transformed remaining interlab devices, created glycerol stocks.