Mid-February 2010:

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GenoCAD workflow and tutorial

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Using the information gathered from analysis of the team wikis and provided design overviews; I attempted to recreate the designs using GenoCAD.

<o:p> </o:p>

First priority was the creation of a comprehensive, exhaustive list of the parts that would be required for each system. Each entry contains the parts name, Biobrick part #, the part type, pertinent data for regulatory parts and links to sequence data and other supplementary data.

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University Groningen<o:p></o:p>

 <o:p></o:p>

<a href="http://2008.igem.org/Team:Groningen">http://2008.igem.org/Team:Groningen</a><o:p></o:p>

 <o:p></o:p>

 <o:p></o:p>

 <o:p></o:p>

Part Name<o:p></o:p>

Part #<o:p></o:p>

Part Type<o:p></o:p>

Length<o:p></o:p>

Strength/efficiency<o:p></o:p>

Sequence and supplementary info<o:p></o:p>

TetR<o:p></o:p>

R0040<o:p></o:p>

Promoter<o:p></o:p>

54<o:p></o:p>

 <o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_R0040">http://partsregistry.org/Part:BBa_R0040</a><o:p></o:p>

PLuxR<o:p></o:p>

R0065<o:p></o:p>

Promoter<o:p></o:p>

97<o:p></o:p>

 <o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_R0065">http://partsregistry.org/Part:BBa_R0065</a><o:p></o:p>

mLuxR<o:p></o:p>

?<o:p></o:p>

Promoter<o:p></o:p>

 <o:p></o:p>

 <o:p></o:p>

N/A<o:p></o:p>

GFP<o:p></o:p>

E0040<o:p></o:p>

Protein<o:p></o:p>

720<o:p></o:p>

N/A<o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_E0040:Design">http://partsregistry.org/Part:BBa_E0040:Design</a><o:p></o:p>

LuxI<o:p></o:p>

C0061<o:p></o:p>

Protein<o:p></o:p>

618<o:p></o:p>

N/A<o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_C0061">http://partsregistry.org/Part:BBa_C0061</a><o:p></o:p>

LuxR<o:p></o:p>

C0062<o:p></o:p>

Protein<o:p></o:p>

756<o:p></o:p>

N/A<o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_C0062">http://partsregistry.org/Part:BBa_C0062</a><o:p></o:p>

aiiA<o:p></o:p>

C0060<o:p></o:p>

Protein<o:p></o:p>

786<o:p></o:p>

N/A<o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_C0060">http://partsregistry.org/Part:BBa_C0060</a><o:p></o:p>

cI<o:p></o:p>

C0051<o:p></o:p>

Protein<o:p></o:p>

 <o:p></o:p>

N/A<o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_C0051">http://partsregistry.org/Part:BBa_C0051</a><o:p></o:p>

B0034<o:p></o:p>

B0034<o:p></o:p>

Ribosome binding site<o:p></o:p>

12<o:p></o:p>

1<o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_B0034">http://partsregistry.org/Part:BBa_B0034</a><o:p></o:p>

B0015<o:p></o:p>

B0015<o:p></o:p>

Ribosome binding site<o:p></o:p>

129<o:p></o:p>

0.98<o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_B0015">http://partsregistry.org/Part:BBa_B0015</a><o:p></o:p>

B0031<o:p></o:p>

B0031<o:p></o:p>

Ribosome binding site<o:p></o:p>

14<o:p></o:p>

0.07<o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_B0031">http://partsregistry.org/Part:BBa_B0031</a><o:p></o:p>

 <o:p></o:p>

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ULB Brussels<o:p></o:p>

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<a href="http://2009.igem.org/Team:ULB-Brussels">http://2009.igem.org/Team:ULB-Brussels</a><o:p></o:p>

 <o:p></o:p>

 <o:p></o:p>

 <o:p></o:p>

LacI Promoter<o:p></o:p>

R0011<o:p></o:p>

Promoter<o:p></o:p>

55<o:p></o:p>

 <o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_R0011">http://partsregistry.org/Part:BBa_R0011</a><o:p></o:p>

cI repressed promoter<o:p></o:p>

R0052<o:p></o:p>

Promoter<o:p></o:p>

46<o:p></o:p>

 <o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_R0052">http://partsregistry.org/Part:BBa_R0052</a><o:p></o:p>

HSL/P22 Hybrid promoter<o:p></o:p>

K145150<o:p></o:p>

Promoter<o:p></o:p>

66<o:p></o:p>

 <o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_K145150">http://partsregistry.org/Part:BBa_K145150</a><o:p></o:p>

LuxR/HSL promoter<o:p></o:p>

R0062<o:p></o:p>

Promoter<o:p></o:p>

55<o:p></o:p>

 <o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_R0062">http://partsregistry.org/Part:BBa_R0062</a><o:p></o:p>

HfsG<o:p></o:p>

K196002<o:p></o:p>

Protein<o:p></o:p>

933<o:p></o:p>

 <o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_K196002">http://partsregistry.org/Part:BBa_K196002</a><o:p></o:p>

HfsH<o:p></o:p>

K196003<o:p></o:p>

Protein<o:p></o:p>

777<o:p></o:p>

 <o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_K196003">http://partsregistry.org/Part:BBa_K196003</a><o:p></o:p>

parE<o:p></o:p>

?<o:p></o:p>

Protein<o:p></o:p>

 <o:p></o:p>

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No biobrick part information but supplementary information on its function can be found here    http://www.ncbi.nlm.nih.gov/pubmed/12010492<o:p></o:p>

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University of Edinburgh<o:p></o:p>

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<a href="http://2008.igem.org/Team:Edinburgh">http://2008.igem.org/Team:Edinburgh</a><o:p></o:p>

 <o:p></o:p>

 <o:p></o:p>

 <o:p></o:p>

cenA<o:p></o:p>

K118023<o:p></o:p>

Protein<o:p></o:p>

1353<o:p></o:p>

 <o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_K118023">http://partsregistry.org/Part:BBa_K118023</a><o:p></o:p>

cex<o:p></o:p>

K118022<o:p></o:p>

Protein<o:p></o:p>

1461<o:p></o:p>

 <o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_K118022">http://partsregistry.org/Part:BBa_K118022</a><o:p></o:p>

bgIX<o:p></o:p>

K118028<o:p></o:p>

Protein<o:p></o:p>

2280<o:p></o:p>

 <o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_K118028">http://partsregistry.org/Part:BBa_K118028</a><o:p></o:p>

SU1<o:p></o:p>

?<o:p></o:p>

Protein<o:p></o:p>

Sequence provided<o:p></o:p>

 <o:p></o:p>

<a href="http://2008.igem.org/Team:Edinburgh/SU1">http://2008.igem.org/Team:Edinburgh/SU1</a><o:p></o:p>

ISO2<o:p></o:p>

?<o:p></o:p>

Protein<o:p></o:p>

Sequence provided<o:p></o:p>

 <o:p></o:p>

<a href="http://2008.igem.org/Team:Edinburgh/ISO2">http://2008.igem.org/Team:Edinburgh/ISO2</a><o:p></o:p>

GBSS<o:p></o:p>

?<o:p></o:p>

Protein<o:p></o:p>

Sequence provided<o:p></o:p>

 <o:p></o:p>

<a href="http://2008.igem.org/Team:Edinburgh/GBS1">http://2008.igem.org/Team:Edinburgh/GBS1</a><o:p></o:p>

glgC<o:p></o:p>

K118015/K118016<o:p></o:p>

Protein<o:p></o:p>

1299<o:p></o:p>

 <o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_K118015">http://partsregistry.org/Part:BBa_K118015</a><o:p></o:p>

E <o:p></o:p>

?<o:p></o:p>

Protein<o:p></o:p>

 <o:p></o:p>

 <o:p></o:p>

Not an official biobrick part - supplementary information can be found here  http://jb.asm.org/cgi/content/abstract/171/8/4334<o:p></o:p>

comK<o:p></o:p>

?<o:p></o:p>

Protein<o:p></o:p>

 <o:p></o:p>

 <o:p></o:p>

Not an official biobrick part - supplementary information can be found here http://www3.davidson.edu/cms/Documents/Academics/Departments/InterdisciplinaryStudies/SimpsonSamanthaThesisOutline12-08pdf.pdf<o:p></o:p>

PcstA<o:p></o:p>

K118011<o:p></o:p>

Promoter<o:p></o:p>

131<o:p></o:p>

 <o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_K118011">http://partsregistry.org/Part:BBa_K118011</a><o:p></o:p>

PcomK<o:p></o:p>

 <o:p></o:p>

Promoter<o:p></o:p>

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Not an official biobrick part <o:p></o:p>

Plac<o:p></o:p>

J33207<o:p></o:p>

Promoter<o:p></o:p>

600<o:p></o:p>

 <o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_J33207">http://partsregistry.org/Part:BBa_J33207</a><o:p></o:p>

J15001<o:p></o:p>

J15001<o:p></o:p>

Ribosome Binding Site<o:p></o:p>

10<o:p></o:p>

 <o:p></o:p>

<a href="http://partsregistry.org/Part:BBa_J15001">http://partsregistry.org/Part:BBa_J15001</a><o:p></o:p>

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<o:p> </o:p>

Following this step, was determining the presence of the parts used in these systems in GenoCAD’s database. Any parts not present in the database were created using GenoCAD’s custom part creation tools. From this point a library of the parts required to create these designs using GenoCAD. However, a number of caveats were initially observed with the program.

These included the inability to create multiple plasmids within the same design canvas. Also there is the inability to explicitly indicate relationship between parts comprising a single design. This is in part down to its nature as a sequence design tool predominantly. Also of note is the inability to place a single gene or multiple genes under the control of multiple promoters without user creation of composite parts. The latter issue being a result of the constraints involved in sequence design to prevent less experienced designers making fatal mistakes.

GenoCAD provides a structured system for the design of synthetic sequences. Based on the use of a set of “grammars” and a hierarchical construction workflow, GenoCAD constrains the possible structure of the synthetic sequences generated to a set of structures that could be functional in a biological context.

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The first option presented to the user after the creation of their library of parts is to select the vector and flanking regions for the coding areas of the plasmid corresponding to their synthetic construct. Selection of the vector leads to a number of further options for the make-up of the expression cassette which in turn leads to further options. In essence, the construction metaphor is an inverted tree with an increase in options from top to bottom. After designing the basic structure of their synthetic construct the specific content for each type of part chosen is filled in. This content is what conveys the behavior of the actual system, more so than the structure of the system. 

The output of this tool is two-fold, the sequence of the synthetic system created and a graphical representation of the system created. An example system with a single negative feedback loop was created. GFP would be the primary output of this design, the levels of which are regulated by the levels of co-produced cI repressor protein. cI repressor protein would act on the cI repressor upstream of both the cI protein gene and GFP genes.

<v:shapetype id="_x0000_t75" coordsize="21600,21600" o:spt="75" o:preferrelative="t" path="m@4@5l@4@11@9@11@9@5xe" filled="f" stroked="f"> <v:stroke joinstyle="miter"/> <v:formulas> <v:f eqn="if lineDrawn pixelLineWidth 0"/> <v:f eqn="sum @0 1 0"/> <v:f eqn="sum 0 0 @1"/> <v:f eqn="prod @2 1 2"/> <v:f eqn="prod @3 21600 pixelWidth"/> <v:f eqn="prod @3 21600 pixelHeight"/> <v:f eqn="sum @0 0 1"/> <v:f eqn="prod @6 1 2"/> <v:f eqn="prod @7 21600 pixelWidth"/> <v:f eqn="sum @8 21600 0"/> <v:f eqn="prod @7 21600 pixelHeight"/> <v:f eqn="sum @10 21600 0"/> </v:formulas> <v:path o:extrusionok="f" gradientshapeok="t" o:connecttype="rect"/> <o:lock v:ext="edit" aspectratio="t"/> </v:shapetype><v:shape id="_x0000_i1026" type="#_x0000_t75" alt="image 1.png" style='width:732pt;height:111.75pt;visibility:visible;mso-wrap-style:square'> <v:imagedata src="GenoCAD%20workflow%20and%20tutorial_files/image002.png" o:title="image 1"/> </v:shape>

<o:p> </o:p>

<v:shape id="Picture_x0020_1" o:spid="_x0000_i1025" type="#_x0000_t75" style='width:732pt; height:112.5pt;visibility:visible;mso-wrap-style:square' o:bordertopcolor="black" o:borderleftcolor="black" o:borderbottomcolor="black" o:borderrightcolor="black"> <v:imagedata src="GenoCAD%20workflow%20and%20tutorial_files/image001.png" o:title=""/> <w:bordertop type="single" width="16"/> <w:borderleft type="single" width="16"/> <w:borderbottom type="single" width="16"/> <w:borderright type="single" width="16"/> </v:shape>

Diagram of example system.<o:p></o:p>

<o:p> </o:p>

The sequence corresponding to this construct as provided by GenoCAD can be found below:

{ggatcctaactcgaggttacattgtcgatctgttcatggtgaacagctttaaatgcaccaaaaactcgtaaaagctctgatgtatctatcttttttacaccgttttcatctgtgcatatggacagttttccctttgatatctaacggtgaacagttgttctacttttgtttgttagtcttgatgcttcact
gatagatacaagagccataagaacctcagatccttccgtatttagccagtatgttctctagtgtgaattcgcggccgcttctagagttgacaaacaagatacattgtatgaaaatacaagaaagtttgttgatactagagaaagaggagaaatactagagatgcgtaaaggagaagaact
tttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaa
tgctttgcgagatacccagatcatatgaaacagcatgactttttcaagtactagagaaagaggagaaatactagagatgagcacaaaaaagaaaccattaacacaagagcagcttgaggacgcacgtcgccttaaagcaatttatgaaaaaaagaaaaatgaacttggcttatcccag
gaatctgtcgcagacaagatggggatggggcagtcaggcgttggtgctttatttaatggcatcaatgcattaaatgcttataacgccgcattgcttgcaaaaattctcaaagttagcgttgaagaatttagcccttcaatcgccagagaaatctactagagccaggcatcaaataaaacgaa
aggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatatactagtagcggccgccctgcagg}

<o:p> </o:p>

However, the sequences for the proteins, corresponding to the major functional elements and actuators of the expected behavior of the system are not included in this output. The sequences of regulatory elements are provided however.

<o:p> </o:p>

<o:p> </o:p>

Modelling of the behavior of a system based on known parameters and experimental data on the individual parts/elements of the system is an important part of the design process. GenoCAD was created to aid in the design process and creation of sequences for the chemical synthesis of these constructs, it is less concerned with the ability to test and/or predict functionality of the created systems in vivo. Due to this inability of GenoCAD to model synthetic systems and the need for such capabilities, we shifted our focus to locating more capable software tools in terms of design and modeling.

<o:p> </o:p>

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