GcatWiki - User contributions [en]

MWSU protocols

2016-06-20T20:35:29Z

ToEckdahl:

'''Purification of DNA'''

[[Isolation of Genomic DNA from Bacteria]]

'''PCR'''

[[iPCR]]

[[Gradient/Standard PCR]]

[[Resuspending Oligos]]

[[Davidson_Missouri_W/colony_PCR | Colony PCR]]

[[Template Preparation for RT-qPCR]]

[[New Chaperone PCR]]

[[LongAmp PCR]]

'''Recombinant DNA Production'''

[[Zymo Research Plasmid Minipreps]]

[[Golden Gate Assembly Protocol]]

[[Golden Gate Assembly Single Molecule Protocol]]

[[Pouring an Agarose Gel]]

[[BioBrick Digestions for Fragment and Vector Preparation]]

[[Fragment Purification]]

[[Gibson Assembly]]

[[Direct Synthesis with Overlapping Oligos]]

[[Annealing Oligos for Cloning]]

[[Ethanol Precipitation of Vector DNA]]

[[Reducing Background from Double Digested Vector]]

[[File: PClone_Procedure_for_GCAT_SB_Workshop_2014_new_version.pptx]]

'''Ligation and Transformation'''

[[BioBrick Ligations]]

[[Ligation and Transformation]]

[[Electroporation Transformation]]

[[SOC Protocol for Transformations]]

'''Screening Clones'''

[[Diagnostic RP Digestion for Checking Insert Size]]

[[DNA Sequencing at Iowa State University]]

[[What to do with a new clone]]

'''Measuring Phenotypes'''

[[Measuring Fluorescence in Bacteria]]

[[Camera Settings for Taking Pictures of Plates]]

'''DNA and E coli'''

[[GCAT Library of Quality Parts]]

[[MWSU Freezer Parts]]

'''Working With Bacteria'''

[[Bacterial Media]]

[[Washing Beads]]

[[Cleaning Plate Replication Pads]]

MWSU protocols

2016-06-20T20:32:12Z

ToEckdahl:

== This is a test ==
'''Purification of DNA'''

[[Isolation of Genomic DNA from Bacteria]]

'''PCR'''

[[iPCR]]

[[Gradient/Standard PCR]]

[[Resuspending Oligos]]

[[Davidson_Missouri_W/colony_PCR | Colony PCR]]

[[Template Preparation for RT-qPCR]]

[[New Chaperone PCR]]

[[LongAmp PCR]]

'''Recombinant DNA Production'''

[[Zymo Research Plasmid Minipreps]]

[[Golden Gate Assembly Protocol]]

[[Golden Gate Assembly Single Molecule Protocol]]

[[Pouring an Agarose Gel]]

[[BioBrick Digestions for Fragment and Vector Preparation]]

[[Fragment Purification]]

[[Gibson Assembly]]

[[Direct Synthesis with Overlapping Oligos]]

[[Annealing Oligos for Cloning]]

[[Ethanol Precipitation of Vector DNA]]

[[Reducing Background from Double Digested Vector]]

[[File: PClone_Procedure_for_GCAT_SB_Workshop_2014_new_version.pptx]]

'''Ligation and Transformation'''

[[BioBrick Ligations]]

[[Ligation and Transformation]]

[[Electroporation Transformation]]

[[SOC Protocol for Transformations]]

'''Screening Clones'''

[[Diagnostic RP Digestion for Checking Insert Size]]

[[DNA Sequencing at Iowa State University]]

[[What to do with a new clone]]

'''Measuring Phenotypes'''

[[Measuring Fluorescence in Bacteria]]

[[Camera Settings for Taking Pictures of Plates]]

'''DNA and E coli'''

[[GCAT Library of Quality Parts]]

[[MWSU Freezer Parts]]

'''Working With Bacteria'''

[[Bacterial Media]]

[[Washing Beads]]

[[Cleaning Plate Replication Pads]]

Eckdahl-replication-protocol

2014-06-04T16:31:23Z

ToEckdahl:

==Procedures==

Grow up E. coli from stock overnight

- One batch in amp, one in amp+chlor

- 4 mM caffeine

Prepare stock of .25 g/mL L-arabinose

- Add 2 mL of this stock to every L of LB+agar, for a final concentration of .5 mG/mL

Measure A590 (cell density) using Synergy/Cytation, dilute concentration to 0.1 with same LB+antibiotic+caffeine. Note that antibiotic will be amp for the 4 clones w/o chaperone, amp+chlor for 20 clones with chaperone.

Combine 0.5 mL of each clone.

Spread 50 uL with 15-20 beads onto Tet-only LB plates

- Prepare tetracycline-LB according to MWSU bacterial media protocol.

- Prepare caffeine disks (establish a precise method of preparing and placing disks, to minimize variation)

- Commercial disks (need to have a certain thickness to absorb enough solution)

- 35 uL 40 mM caffeine solution per disk

- Put dry disk on sterile petri dish, add caffeine (filter sterilized) and let sit for 1 minute

- Stab with the smallest sterile needle you have and transfer it to the tet plate, take sterile pipet tip and remove it from the needle.

- Let sit for 5 minutes with lid cracked open under sterile hood

- Incubate upside down

- 37degrees C overnight

- Room temperature for 4 days (with daily evaluation and regular photos and UV box photos)

Pick out each colony with sterile pipet tip and deposit in sterile microtiter plate/pcr tubes (LB+amp colony) filled with 10uL LB+amp. Mix well by pipetting up and down, using the same pipet tip for each colony. Number colonies and record whether each colony is big or little.

Pipet 5 uL of each LB+amp+clone to 195 LB+amp+chlor, using the same pipet tip for the same colony.

Add LB+amp to LB+amp clones to produce 200 uL LB+amp+clones, for each colony.

Incubate and measure A590 to determine how many colonies are still growing.

Measure RFP and GFP fluorescence

Spot each colony on a single plate (2 uL per spot)

Determine chaperone # with PCR; run on agarose gel

New Chaperone PCR Mixture

- 2 uL overnight culture of bacterial clone (picked out of amp+chlor)

- 0.4 uL primer cocktail with new chaperone primers equally mixed from 100 uM stocks

- 7.6 uL water

- 10 uL 2x GoTaq Green

New Chaperone PCR Thermal Profile

- Initial denaturation: 10 minutes at 94 degrees

- 20 cycles of 94 degrees, 15 sec; 51 degrees, 15 sec; 74 degrees, 2 minutes

- Final extension: 74 degrees, 5 minutes

Origin PCR Mixture

- 2 uL overnight culture of bacterial clone (picked out of either amp or amp+chlor, according to the presence of the chaperone),

- 0.5 uL primer cocktail with origin primers equally mixed from 100 uM stocks

- 7.5 uL water

- 10 uL 2x GoTaq Green

Origin PCR Thermal Profile

- Initial denaturation: 94 degrees, 10 minutes

- 20 cycles of touch-down PCR: 94 degrees for 15 sec, 64.5 to 44.5 degrees for 15 sec; 74 degrees for 1 minute

- 20 cycles of PCR: 94 degrees for 15 sec, 44.5 degrees for 15 sec; 74 degrees for 1 minute

- Final extension: 74 degrees for 5 minutes

==Plasmids==

J119346

– High promoter

– High C-dog (RBS)

– RFP

J119347

– Low promoter

– Low C-dog

– GFP

== Origins ==

pSB1A2 High copy number

J119310 Low copy number

Eckdahl-replication-protocol

2014-06-04T16:28:25Z

ToEckdahl:

==Procedures==

Grow up E. coli from stock overnight

- One batch in amp, one in amp+chlor

- 4 mM caffeine

Prepare stock of .25 g/mL L-arabinose

- Add 2 mL of this stock to every L of LB+agar, for a final concentration of .5 mG/mL

Measure A590 (cell density) using Synergy/Cytation, dilute concentration to 0.1 with same LB+antibiotic+caffeine. Note that antibiotic will be amp for the 4 clones w/o chaperone, amp+chlor for 20 clones with chaperone.

Combine 0.5 mL of each clone.

Spread 50 uL with 15-20 beads onto Tet-only LB plates

- Prepare tetracycline-LB according to MWSU bacterial media protocol.

- Prepare caffeine disks (establish a precise method of preparing and placing disks, to minimize variation)

- Commercial disks (need to have a certain thickness to absorb enough solution)

- 35 uL 40 mM caffeine solution per disk

- Put dry disk on sterile petri dish, add caffeine (filter sterilized) and let sit for 1 minute

- Stab with the smallest sterile needle you have and transfer it to the tet plate, take sterile pipet tip and remove it from the needle.

- Let sit for 5 minutes with lid cracked open under sterile hood

- Incubate upside down

- 37degrees C overnight

- Room temperature for 4 days (with daily evaluation and regular photos and UV box photos)

Pick out each colony with sterile pipet tip and deposit in sterile microtiter plate/pcr tubes (LB+amp colony) filled with 10uL LB+amp. Mix well by pipetting up and down, using the same pipet tip for each colony. Number colonies and record whether each colony is big or little.

Pipet 5 uL of each LB+amp+clone to 195 LB+amp+chlor, using the same pipet tip for the same colony.

Add LB+amp to LB+amp clones to produce 200 uL LB+amp+clones, for each colony.

Incubate and measure A590 to determine how many colonies are still growing.

Measure RFP and GFP fluorescence

Spot each colony on a single plate (2 uL per spot)

Determine chaperone # with PCR; run on agarose gel

New Chaperone PCR Mixture

- 2 uL overnight culture of bacterial clone (picked out of amp+chlor)

- 0.4 uL primer cocktail with new chaperone primers equally mixed from 100 uM stocks

- 7.6 uL water

- 10 uL 2x GoTaq Green

New Chaperone PCR Thermal Profile

- Initial denaturation: 10 minutes at 94 degrees

- 20 cycles of 94 degrees, 15 sec; 51 degrees, 15 sec; 74 degrees, 2 minutes

- Final extension: 74 degrees, 5 minutes

Origin PCR Mixture

- 2 uL overnight culture of bacterial clone (picked out of either amp or amp+chlor, according to the presence of the chaperone),

- 0.5 uL primer cocktail with origin primers equally mixed from 100 uM stocks

- 7.5 uL water

- 10 uL 2x GoTaq Green

Origin PCR Thermal Profile

- Initial denaturation: 94 degrees, 10 minutes

- 20 cycles of touch-down PCR: 94 degrees for 15 sec, 64.5 to 44.5 degrees for 15 sec; 74 degrees for 1 minute

- 20 cycles of PCR: 94 degrees for 15 sec, 44.5 degrees for 15 sec; 74 degrees for 1 minute

- Final extension: 74 degrees for 5 minutes

==Plamids==

J119346

– High promoter

– High C-dog (RBS)

– RFP

J119347

– Low promoter

– Low C-dog

– GFP

== Origins ==

pSB1A2 High copy number

J119310 Low copy number

Eckdahl-replication-protocol

2014-06-04T16:27:32Z

ToEckdahl:

==Procedures==

Grow up E. coli from stock overnight

- One batch in amp, one in amp+chlor

- 4 mM caffeine

Prepare stock of .25 g/mL L-arabinose

- Add 2 mL of this stock to every L of LB+agar, for a final concentration of .5 mG/mL

Measure A590 (cell density) using Synergy/Cytation, dilute concentration to 0.1 with same LB+antibiotic+caffeine. Note that antibiotic will be amp for the 4 clones w/o chaperone, amp+chlor for 20 clones with chaperone.

Combine 0.5 mL of each clone.

Spread 50 uL with 15-20 beads onto Tet-only LB plates

- Prepare tetracycline-LB according to MWSU bacterial media protocol.

- Prepare caffeine disks (establish a precise method of preparing and placing disks, to minimize variation)

- Commercial disks (need to have a certain thickness to absorb enough solution)

- 35 uL 40 mM caffeine solution per disk

- Put dry disk on sterile petri dish, add caffeine (filter sterilized) and let sit for 1 minute

- Stab with the smallest sterile needle you have and transfer it to the tet plate, take sterile pipet tip and remove it from the needle.

- Let sit for 5 minutes with lid cracked open under sterile hood

- Incubate upside down

- 37degrees C overnight

- Room temperature for 4 days (with daily evaluation and regular photos and UV box photos)

Pick out each colony with sterile pipet tip and deposit in sterile microtiter plate/pcr tubes (LB+amp colony) filled with 10uL LB+amp. Mix well by pipetting up and down, using the same pipet tip for each colony. Number colonies and record whether each colony is big or little.

Pipet 5 uL of each LB+amp+clone to 195 LB+amp+chlor, using the same pipet tip for the same colony.

Add LB+amp to LB+amp clones to produce 200 uL LB+amp+clones, for each colony.

Incubate and measure A590 to determine how many colonies are still growing.

Measure RFP and GFP fluorescence

Spot each colony on a single plate (2 uL per spot)

Determine chaperone # with PCR; run on agarose gel

New Chaperone PCR Mixture

- 2 uL overnight culture of bacterial clone (picked out of amp+chlor)

- 0.4 uL primer cocktail with origin primers equally mixed from 100 uM stocks

- 7.6 uL water

- 10 uL 2x GoTaq Green

New Chaperone PCR Thermal Profile

- Initial denaturation: 10 minutes at 94 degrees

- 20 cycles of 94 degrees, 15 sec; 51 degrees, 15 sec; 74 degrees, 2 minutes

- Final extension: 74 degrees, 5 minutes

Origin PCR Mixture

- 2 uL overnight culture of bacterial clone (picked out of either amp or amp+chlor, according to the presence of the chaperone),

- 0.5 uL primer cocktail with origin primers equally mixed from 100 uM stocks

- 7.5 uL water

- 10 uL 2x GoTaq Green

Origin PCR Thermal Profile

- Initial denaturation: 94 degrees, 10 minutes

- 20 cycles of touch-down PCR: 94 degrees for 15 sec, 64.5 to 44.5 degrees for 15 sec; 74 degrees for 1 minute

- 20 cycles of PCR: 94 degrees for 15 sec, 44.5 degrees for 15 sec; 74 degrees for 1 minute

- Final extension: 74 degrees for 5 minutes

==Plamids==

J119346

– High promoter

– High C-dog (RBS)

– RFP

J119347

– Low promoter

– Low C-dog

– GFP

== Origins ==

pSB1A2 High copy number

J119310 Low copy number

Eckdahl-replication-protocol

2014-06-04T16:26:50Z

ToEckdahl:

==Procedures==

Grow up E. coli from stock overnight

- One batch in amp, one in amp+chlor

- 4 mM caffeine

Prepare stock of .25 g/mL L-arabinose

- Add 2 mL of this stock to every L of LB+agar, for a final concentration of .5 mG/mL

Measure A590 (cell density) using Synergy/Cytation, dilute concentration to 0.1 with same LB+antibiotic+caffeine. Note that antibiotic will be amp for the 4 clones w/o chaperone, amp+chlor for 20 clones with chaperone.

Combine 0.5 mL of each clone.

Spread 50 uL with 15-20 beads onto Tet-only LB plates

- Prepare tetracycline-LB according to MWSU bacterial media protocol.

- Prepare caffeine disks (establish a precise method of preparing and placing disks, to minimize variation)

- Commercial disks (need to have a certain thickness to absorb enough solution)

- 35 uL 40 mM caffeine solution per disk

- Put dry disk on sterile petri dish, add caffeine (filter sterilized) and let sit for 1 minute

- Stab with the smallest sterile needle you have and transfer it to the tet plate, take sterile pipet tip and remove it from the needle.

- Let sit for 5 minutes with lid cracked open under sterile hood

- Incubate upside down

- 37degrees C overnight

- Room temperature for 4 days (with daily evaluation and regular photos and UV box photos)

Pick out each colony with sterile pipet tip and deposit in sterile microtiter plate/pcr tubes (LB+amp colony) filled with 10uL LB+amp. Mix well by pipetting up and down, using the same pipet tip for each colony. Number colonies and record whether each colony is big or little.

Pipet 5 uL of each LB+amp+clone to 195 LB+amp+chlor, using the same pipet tip for the same colony.

Add LB+amp to LB+amp clones to produce 200 uL LB+amp+clones, for each colony.

Incubate and measure A590 to determine how many colonies are still growing.

Measure RFP and GFP fluorescence

Spot each colony on a single plate (2 uL per spot)

Determine chaperone # with PCR; run on agarose gel

New Chaperone PCR Mixture

- 2 uL overnight culture of bacterial clone (picked out of amp+chlor)

- 0.4 uL primer cocktail with origin primers equally mixed from 100 uM stocks

- 7.6 uL water

- 10 uL 2x GoTaq Gree

New Chaperone PCR Thermal Profile

- Initial denaturation: 10 minutes at 94 degrees

- 20 cycles of 94 degrees, 15 sec; 51 degrees, 15 sec; 74 degrees, 2 minutes

- Final extension: 74 degrees, 5 minutes

Determine origin with PCR; run on agarose gel

Origin PCR Mixture

- 2 uL overnight culture of bacterial clone (picked out of either amp or amp+chlor, according to the presence of the chaperone),

- 0.5 uL primer cocktail with origin primers equally mixed from 100 uM stocks

- 7.5 uL water

- 10 uL 2x GoTaq Green

Origin PCR Thermal Profile

- Initial denaturation: 94 degrees, 10 minutes

- 20 cycles of touch-down PCR: 94 degrees for 15 sec, 64.5 to 44.5 degrees for 15 sec; 74 degrees for 1 minute

- 20 cycles of PCR: 94 degrees for 15 sec, 44.5 degrees for 15 sec; 74 degrees for 1 minute

- Final extension: 74 degrees for 5 minutes

==Plamids==

J119346

– High promoter

– High C-dog (RBS)

– RFP

J119347

– Low promoter

– Low C-dog

– GFP

== Origins ==

pSB1A2 High copy number

J119310 Low copy number

Eckdahl-replication-protocol

2014-06-04T16:17:40Z

ToEckdahl:

==Procedures==

Grow up E. coli from stock overnight

- One batch in amp, one in amp+chlor

- 4 mM caffeine

Prepare stock of .25 g/mL L-arabinose

- Add 2 mL of this stock to every L of LB+agar, for a final concentration of .5 mG/mL

Measure A590 (cell density) using Synergy/Cytation, dilute concentration to 0.1 with same LB+antibiotic+caffeine. Note that antibiotic will be amp for the 4 clones w/o chaperone, amp+chlor for 20 clones with chaperone.

Combine 0.5 mL of each clone.

Spread 50 uL with 15-20 beads onto Tet-only LB plates

- Prepare tetracycline-LB according to MWSU bacterial media protocol.

- Prepare caffeine disks (establish a precise method of preparing and placing disks, to minimize variation)

- Commercial disks (need to have a certain thickness to absorb enough solution)

- 35 uL 40 mM caffeine solution per disk

- Put dry disk on sterile petri dish, add caffeine (filter sterilized) and let sit for 1 minute

- Stab with the smallest sterile needle you have and transfer it to the tet plate, take sterile pipet tip and remove it from the needle.

- Let sit for 5 minutes with lid cracked open under sterile hood

- Incubate upside down

- 37degrees C overnight

- Room temperature for 4 days (with daily evaluation and regular photos and UV box photos)

Pick out each colony with sterile pipet tip and deposit in sterile microtiter plate/pcr tubes (LB+amp colony) filled with 10uL LB+amp. Mix well by pipetting up and down, using the same pipet tip for each colony. Number colonies and record whether each colony is big or little.

Pipet 5 uL of each LB+amp+clone to 195 LB+amp+chlor, using the same pipet tip for the same colony.

Add LB+amp to LB+amp clones to produce 200 uL LB+amp+clones, for each colony.

Incubate and measure A590 to determine how many colonies are still growing.

Measure RFP and GFP fluorescence

Spot each colony on a single plate (2 uL per spot)

Determine chaperone # with PCR; run on agarose gel

Mixture

2 uL overnight culture of bacterial clone (picked out of either amp or amp+chlor, according to the presence of the chaperone),
primer cocktail with chaperone primers

water

2x GoTaq Green

PCR Steps

- Initial denaturation: 10 minutes at 94 degrees

- 30 cycles of 94 degrees, 15 sec; 46 degrees, 15 sec; 74 degrees, 6 minutes

- Final extension: 74 degrees, 5 minutes

Determine origin with PCR; run on agarose gel

Mixture

- 2 uL overnight culture of bacterial clone (picked out of either amp or amp+chlor, according to the presence of the chaperone),

- 0.4 uL primer cocktail with origin primers 100 uM each

- 7.6 uL water

- 10 uL 2x GoTaq Green

PCR Steps

- Initial denaturation: 94 degrees, 10 minutes

- 20 cycles of touch-down PCR: 94 degrees for 15 sec, 64.5 to 44.5 degrees for 15 sec; 74 degrees for 1 minute

- 20 cycles of PCR: 94 degrees for 15 sec, 44.5 degrees for 15 sec; 74 degrees for 1 minute

- Final extension: 74 degrees for 5 minutes

==Plamids==

J119346

– High promoter

– High C-dog (RBS)

– RFP

J119347

– Low promoter

– Low C-dog

– GFP

== Origins ==

pSB1A2 High copy number

J119310 Low copy number

Rational Design of Riboswitches: papers to read

2014-05-30T14:26:34Z

ToEckdahl:

Wachsmuth, et al - [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575828/ De novo design of a synthetic riboswitch that regulates transcription termination]
* engineered switch terminates '''transcription''' in the absence of theophylline (6.5 fold induction in presence of theophylline)
* model folding of many candidate switches in steps of 5-10 nt (to simulate different elongation speeds) using RNAfold
* find distribution of MFE in shuffled sequences to characterize those structures more/less stable than expected by chance

Beisel and Smolke - [http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.1000363 Design Principles for Riboswitch Function]
* In this paper they develop a kinetics model for riboswitches that function in any of three ways: translational repression, transcriptional termination, and mRNA destabilization. The mathematical modelling involved differential equations to describe each step in the mechanism of transcription, ligand binding, translation, and RNA degradation. They emphasized the importance of the relative rates of reversible steps such as RNA folding and ligand binding to irreversible steps such as transcription termination and RNA degradation. They built theophylline riboswitches to confirm important aspects of their model.
Dixon, et al - [http://www.pnas.org/content/107/7/2830.full#ref-19 Reengineering Orthogonally Selective Riboswitches]

Rieder, et al (referenced by Dixon)- [http://onlinelibrary.wiley.com/doi/10.1002/cbic.200700057/full Ligand-Induced Folding of the Adenosine Deaminase A-Riboswitch and Implications on Riboswitch Translational Control]

Perdizet, et al - [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3295289/ Transcriptional pausing coordinates folding of the aptamer domain and the expression platform of a riboswitch]

Ceres, et al (Mar 2013) - [http://pubs.acs.org/doi/ipdf/10.1021/sb4000096 Modularity of Select Riboswitch Expression Platforms Enables Facile Engineering of Novel Genetic Regulatory Devices]
* OFF switches

Ceres, Trausch and Batey (Dec 2013) - [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3905868/ Engineering modular ‘ON’ RNA switches using biological components]

MWSU protocols

2014-05-30T12:55:25Z

ToEckdahl:

'''Purification of DNA'''
[[Isolation of Genomic DNA from Bacteria]]

'''PCR'''

[[iPCR]]

[[Standard PCR]]

[[Resuspending Oligos]]

[[Davidson_Missouri_W/colony_PCR | Colony PCR]]

[[Template Preparation for RT-qPCR]]

[[New Chaperone PCR]]

'''Recombinant DNA Production'''

[[Zymo Research Plasmid Minipreps]]

[[Golden Gate Assembly Protocol for BsmB1]]

[[Pouring an Agarose Gel]]

[[BioBrick Digestions for Fragment and Vector Preparation]]

[[Fragment Purification]]

[[Gibson Assembly]]

[[Direct Synthesis with Overlapping Oligos]]

[[Annealing Oligos for Cloning]]

[[Ethanol Precipitation of Vector DNA]]

[[Reducing Background from Double Digested Vector]]

[[File: PClone_Procedure_for_GCAT_SB_Workshop_2014_new_version.pptx]]

'''Ligation and Transformation'''

[[BioBrick Ligations]]

[[Ligation and Transformation]]

[[Electroporation Transformation]]

[[Bacterial Media]]

'''Screening Clones'''

[[Diagnostic RP Digestion for Checking Insert Size]]

[[DNA Sequencing at Iowa State University]]

[[What to do with a new clone]]

'''Measuring Phenotypes'''

[[Measuring Fluorescence in Bacteria]]

'''DNA and E coli'''

[[GCAT Library of Quality Parts]]

[[MWSU Freezer Parts]]

File:PClone Procedure for GCAT SB Workshop 2014 new version.pptx

2014-05-30T12:54:26Z

ToEckdahl:

Summer 2014 SynBio Project (Davidson and MWSU)

2014-05-29T16:41:04Z

ToEckdahl:

=== Presentations during MWSU visit to Davidson ===

Central Dogma presentation [[File:Central_dogma.pptx]]

PCR presentation [[File:PCR.pptx]]

Cloning presentation [[File:Cloning.pptx]]

Riboswitch presentation [[File:Riboswitch_function.pptx]]

Agent based modeling presentation [[File:AgentBased_Modeling.pptx]]

Competition presentation [[File:Competition_Modeling.xlsx]]

Programmed evolution presentation [[File:Programmed_Evolution.pdf]]

Caffeine results [[File:Caffeine_Disk.pptx]]

Ammeline presentation [[File:Ammeline.pptx]]

Repressilator modeling exercises [[File:repressilator_modeling.docx]]

Repressilator modeling Excel file [[File:Repressilator_model.xls]]

Repressilator modeling Netlogo file [[File:Repressilator_mod.nlogo.zip]]

=== Biology Files ===

Chaperone plasmid DNA sequences [[File:Chaperone_plasmid_DNA_sequences.docx]]

Origin PCR Gels 5-26-14[[File:5-26-14 Orig PCR Elecpho Gel Pic.doc]]

Chaperone PCR and Repeat Origin PCR 5-27-14[[File:5-27-14_Chap_PCR_and_Repeat_Ori_PCR.doc]]

Chaperone PCR of original clones 5-27-14[[File:5-27-14_chaperone_pcr_clones_1-24.doc]]

Origin PCR of original clones 5-27-14[[File:5-27-14 Ori PCR 1-24.doc ]]

New Chap PCR clones 1-5[[File:5-28-14_New_Chap_PCR_1-5.doc ]]

Combinations Labeling System [[File:Labeling.xlsx]]

ThyA Fitness Module [[File:ThyA fitness module.docx]]

===Sub-pages on various topics===
'''
[[MATH]]
'''

[[Catherine Doyle Thesis Materials]]

[[Repeating 20 Clone Experiments]]

[[Extending theophylline application]]

[[Melamine iteration]]

[[Ramping up Programmed Evolution]]

[[Rational Design of Riboswitches: papers to read]]

Bacterial Media

2014-05-28T14:01:01Z

ToEckdahl:

'''Missouri Western Synthetic Biology Protocols
Bacterial Media Prep'''

'''LB Broth'''

5 g Yeast Extract
10 g Tryptone
5 g NaCl

QSV 1 liter

'''LB Plates''' (makes about 35 plates)

5 g Yeast Extract
10 g Tryptone
5 g NaCl
15 g BactoAgar

QSV 1 liter

'''Plate Codes'''

#Black line only LB none
#Black line, Red line LB Amp
#Black line, Blue line LB Tet
#Black line, Green line LB Kan
#Black line, Cyan line LB Chlor

'''Ampicillin'''
Stock – 50 mg/ml

Weigh out 2.5 g Ampicillin, sodium salt (Sigma A0166)
Dissolve in 25 ml dH20
Add 25 ml Ethanol

Working concentration 50 ug/ml (1 ml stock solution per liter)

'''Tetracycline'''
Stock – 5 mg/ml

Weigh out 0.25 g Tetracycline HCl (Sigma T7660)

Dissolve in 50 ml 70% Ethanol

Mix/vortex vigorously

Filter sterilize

Make 4 ml aliquot in foil covered 15 ml tubes

Working concentration 20 ug/ml (4 ml stock solution per liter)

Light Sensitive! Keep plates in darkness

'''Kanamycin'''
Stock – 10 mg/ml

Weigh out 0.5 g Kanamycin sulfate (Sigma K1377)
Dissolve in 50 ml dH20

Working concentration 20 ug/ml (2 ml stock solution per liter)

'''Chloramphenicol'''
Stock – 35 mg/ml

Weigh out 1.75 g Chloramphenicol (Sigma C0378)
Add 50 ml 100% Ethanol
Mix/vortex vigorously

Working concentration 35 ug/ml (1 ml stock solution per liter)

'''L-Arabinose'''
Stock – 250 mg/ml

Weigh out 2.5 g L-Arabinose
Add 10 ml dH2O
Mix/vortex vigorously

Working concentration 0.5 mg/ml (2 ml stock solution per liter)

Summer 2014 SynBio Project (Davidson and MWSU)

2014-05-21T18:37:56Z

ToEckdahl:

Central Dogma presentation [[File:Central_dogma.pptx]]

PCR presentation [[File:PCR.pptx]]

Cloning presentation [[File:Cloning.pptx]]

Riboswitch presentation [[File:Riboswitch_function.pptx]]

Agent based modeling presentation [[File:AgentBased_Modeling.pptx]]

Competition presentation [[File:Competition_Modeling.xlsx]]

Programmed evolution presentation [[File:Programmed_Evolution.pdf]]

Caffeine results [[File:Caffeine_Disk.pptx]]

Ammeline presentation [[File:Ammeline.pptx]]

Repressilator modeling exercises [[File:repressilator_modeling.docx]]

Repressilator modeling Excel file [[File:Repressilator_model.xls]]

Repressilator modeling Netlogo file [[File:Repressilator_mod.nlogo.zip]]

Chaperone plasmid DNA sequences [[File:Chaperone_plasmid_DNA_sequences.docx]]

'''
[[MATH]]
'''

[[Catherine Doyle Thesis Materials]]

[[Repeating 20 Clone Experiments]]

[[Extending theophylline application]]

[[Melamine iteration]]

[[Ramping up Programmed Evolution]]

File:Chaperone plasmid DNA sequences.docx

2014-05-21T18:37:21Z

ToEckdahl:

Summer 2014 SynBio Project (Davidson and MWSU)

2014-05-21T18:36:04Z

ToEckdahl:

Central Dogma presentation [[File:Central_dogma.pptx]]

PCR presentation [[File:PCR.pptx]]

Cloning presentation [[File:Cloning.pptx]]

Riboswitch presentation [[File:Riboswitch_function.pptx]]

Agent based modeling presentation [[File:AgentBased_Modeling.pptx]]

Competition presentation [[File:Competition_Modeling.xlsx]]

Programmed evolution presentation [[File:Programmed_Evolution.pdf]]

Caffeine results [[File:Caffeine_Disk.pptx]]

Ammeline presentation [[File:Ammeline.pptx]]

Repressilator modeling exercises [[File:repressilator_modeling.docx]]

Repressilator modeling Excel file [[File:Repressilator_model.xls]]

Repressilator modeling Netlogo file [[File:Repressilator_mod.nlogo.zip]]

Chaperone plasmid DNA sequences [[File:Chaperone_plasmid_DNA_sequences_5-21-14.docx]]

'''
[[MATH]]
'''

[[Catherine Doyle Thesis Materials]]

[[Repeating 20 Clone Experiments]]

[[Extending theophylline application]]

[[Melamine iteration]]

[[Ramping up Programmed Evolution]]

Summer 2014 SynBio Project (Davidson and MWSU)

2014-05-21T18:35:41Z

ToEckdahl:

Central Dogma presentation [[File:Central_dogma.pptx]]

PCR presentation [[File:PCR.pptx]]

Cloning presentation [[File:Cloning.pptx]]

Riboswitch presentation [[File:Riboswitch_function.pptx]]

Agent based modeling presentation [[File:AgentBased_Modeling.pptx]]

Competition presentation [[File:Competition_Modeling.xlsx]]

Programmed evolution presentation [[File:Programmed_Evolution.pdf]]

Caffeine results [[File:Caffeine_Disk.pptx]]

Ammeline presentation [[File:Ammeline.pptx]]

Repressilator modeling exercises [[File:repressilator_modeling.docx]]

Repressilator modeling Excel file [[File:Repressilator_model.xls]]

Repressilator modeling Netlogo file [[File:Repressilator_mod.nlogo.zip]]

Chaperone plasmid DNA sequences [[Chaperone_plasmid_DNA_sequences_5-21-14.docx]]

'''
[[MATH]]
'''

[[Catherine Doyle Thesis Materials]]

[[Repeating 20 Clone Experiments]]

[[Extending theophylline application]]

[[Melamine iteration]]

[[Ramping up Programmed Evolution]]

Dream Gene

2013-07-26T14:35:20Z

ToEckdahl:

'''Dream Gene Characteristics'''

Small (less than 1,200 base pairs) 
Measurable Output (Riboswitch, Color) 
Functions in E. coli, but orthogonal 
Already Studied/or not 
Beneficial 
Pharmaceutical 
Biofuel 
Chemical Commodities 
Bioremediation 

Upload documents here.

[[File:Dream_Gene_July_22nd_2013_Claire_Shinneman.pptx]]

[[File:msb_gene.docx]]

[[File:Interleukin 6.docx]]

[[File:Glycerol_dehydrogenase_.docx‎]]

File:Dream Gene July 22nd 2013 Claire Shinneman.pptx

2013-07-26T14:34:31Z

ToEckdahl: ToEckdahl uploaded a new version of "File:Dream Gene July 22nd 2013 Claire Shinneman.pptx"

File:Dream Gene July 22nd 2013 Claire Shinneman.pptx

2013-07-26T14:32:57Z

ToEckdahl:

Dream Gene

2013-07-26T14:28:41Z

ToEckdahl:

'''Dream Gene Characteristics'''

Small (less than 1,200 base pairs) 
Measurable Output (Riboswitch, Color) 
Functions in E. coli, but orthogonal 
Already Studied/or not 
Beneficial 
Pharmaceutical 
Biofuel 
Chemical Commodities 
Bioremediation 

Upload documents here.

[[File:msb_gene.docx]]

[[File:Interleukin 6.docx]]

[[File:Glycerol_dehydrogenase_.docx‎]]

Dream Gene

2013-07-26T14:27:47Z

ToEckdahl:

'''Dream Gene Characteristics'''

Small (less than 1,200 base pairs)
Measurable Output (Riboswitch, Color)
Functions in E. coli, but orthogonal
Already Studied/or not
Beneficial
Pharmaceutical
Biofuel
Chemical Commodities
Bioremediation

Upload documents here.

[[File:msb_gene.docx]]

[[File:Interleukin 6.docx]]

[[File:Glycerol_dehydrogenase_.docx‎]]

Biology

2013-07-26T14:25:33Z

ToEckdahl:

[[Davidson Protocols]] 
[[MWSU_protocols]] 

[[J-GGA Scaffold Design]]

[[Origin of Replication]]

[[File:Gelpouring.jpg|200px|thumb|right|Look, Mom, no EtBr!]]

[//gcat.davidson.edu/SynBio13/primer GGA Primer Designer]

'''Topics to Investigate'''

'''Family of Xanthine compounds'''

[[File:Caffeine_demethylase_word_doc.docx]]

High Priority = more people

* '''Phoebe and Claire:''' eCDM8 working to make theophylline with biosensor and fitness modules (TetR) to report out. 
Which plasmid carries which device? 
Biosensor 
Fitness (tetA) 
feeds into stress module 
* '''Sachii and Jess:''' Determine the junction sequences and associated primers 
(use existing rules) 
build scaffold for insert segments 
include orI (collaborate with copy number research) 
include drug resistance gene 
test JGG design 
* '''Spencer and Sara:''' test fitness module with adhE
compare with tetA 
fine tune tetA resistance 
combine with tetA 
Does theophylline diffuse across the membrane and thus the system fails? 
* '''Brandon and Erich:''' Copy number of plasmids 
Can we alter Ori to produce a range of plasmid densities in ''E. coli''? 

Second Order Priority = one person
* produce different CDM alleles
* swap out different components (promoters, RBS, alleles)
* test out programmed evolution
* Stress module needs more work (see eCDM8 outputs)

File:Caffeine demethylase word doc.docx

2013-07-26T14:24:06Z

ToEckdahl:

Biology

2013-07-26T14:23:25Z

ToEckdahl:

[[Davidson Protocols]] 
[[MWSU_protocols]] 

[[J-GGA Scaffold Design]]

[[Origin of Replication]]

[[File:Gelpouring.jpg|200px|thumb|right|Look, Mom, no EtBr!]]

[//gcat.davidson.edu/SynBio13/primer GGA Primer Designer]

'''Topics to Investigate'''

'''Family of Xanthine compounds'''

High Priority = more people

* '''Phoebe and Claire:''' eCDM8 working to make theophylline with biosensor and fitness modules (TetR) to report out. 
Which plasmid carries which device? 
Biosensor 
Fitness (tetA) 
feeds into stress module 
* '''Sachii and Jess:''' Determine the junction sequences and associated primers 
(use existing rules) 
build scaffold for insert segments 
include orI (collaborate with copy number research) 
include drug resistance gene 
test JGG design 
* '''Spencer and Sara:''' test fitness module with adhE
compare with tetA 
fine tune tetA resistance 
combine with tetA 
Does theophylline diffuse across the membrane and thus the system fails? 
* '''Brandon and Erich:''' Copy number of plasmids 
Can we alter Ori to produce a range of plasmid densities in ''E. coli''? 

Second Order Priority = one person
* produce different CDM alleles
* swap out different components (promoters, RBS, alleles)
* test out programmed evolution
* Stress module needs more work (see eCDM8 outputs)

Annealing Oligos for Cloning

2013-07-04T14:28:46Z

ToEckdahl:

'''Anneal Oligos'''

# Design oligos (the [http://gcat.davidson.edu/igem10/ Oligator] is a useful tool for this)
# Order oligos at 100 uM concentration (eg. [http://www.idtdna.com/Home/Home.aspx IDT])
# Set up annealing reaction

2 ul 10X annealing buffer (1 M NaCl, 100 mM Tris-HCl pH 7.4)
1 ul each oligo (5 uM final concentration)
dH2O to 20 ul total

# Boil 4 minutes in beaker with 400 ml H2O
# Turn off heat and let cool at least 2 hours

'''Dilute Oligos for Ligation'''

Calculate the molarity of the vector that will be used in the ligation (assuming you will use 1 ul of the vector)

(__ ng vector)/(670 ng/nmol-base pair x __ base pairs x 10 exp-6 L) = ___ nM (nmol/L)

Example: (50 ng)/(670 x 2079 bp x 10 exp-6) = 36 nM

Calculate the molarity of annealed oligos to be used for the ligation (assuming your will use 1 ul of the diluted oligos)

__ nM vector x (molar excess of oligos)

Ligation Example: 36 nM x 3 = 108 nM oligos

GGA Example: 36 nM x 1 = 36 nM oligos

Calculate how much to dilute annealed oligos

Stock oligos = 100 uM
Annealed oligos = 5 uM = 5000 nM
Dilution factor for oligos = 5000 nM / desired oligo molarity

Ligation Example: 5000 nM /108 nM = 46 dilution factor

GGA Example: 5000 nM /36 nM = 139 dilution factor

Dilute annealed oligos with dH20

Ligation Example: 1 ul annealed oligos plus 45 ul dH20

GGA Example: 1 ul annealed oligos plus 138 ul dH20

Use 1 ul of vector and 1 ul of diluted oligos for ligation

Main Page

2013-06-19T21:21:20Z

ToEckdahl:

==[[Davidson Protocols]]==
 

==[[MWSU_protocols]]==
 

 
==[[Summer 2013 SynBio Project (Davidson and MWSU)]]==
 

==[[College Merit Aid]]==

 
==[[iRobot Energy Saver Project]]==

 
==[[Summer 2012 SynBio Project (Davidson and MWSU)]]==

 
==[[Synthetic Biology Network Research]]==

 
==[[Genome Assembly Project: Leland Taylor '12]]==
 

==[[Blueberry Genome Project for Bio343]]==

 

== [[Halomicrobium mukohataei Genome Fall 2009]] ==

 
== [[Halorhabdus utahensis Genome]] ==

 
==[[Network Research with Synthetic Biology]]==

 
== [[Missouri Western/Davidson SynBio 2011]] ==

 
== [[Missouri Western/Davidson iGEM2010]] ==

 
== [[Missouri Western/Davidson iGEM2009]] ==

 
== [[Davidson/Missouri Western iGEM2008]] ==

 
== [[MAGIC Tool Development]] ==

 
== [[Nova Southeastern University]] ==

 

== [[Davidson College]]== Small liberal arts college near Charlotte, NC.
A. Malcolm Campbell and Laurie J. Heyer GCAT faculty

* [http://gcat.davidson.edu/GcatWiki/index.php/User:Kahaynes Karmella A. Haynes], Visiting Assitant Professor of Biology

* [[A_Review_of_Synthetic_Biology |A Review of Synthetic Biology]] - Davidson College Synthetic Biology Seminar (Fall 2007)

* [[Laboratory Notebooks]]

* [[2009-2010 Biology Curriculum Wiki]]

* [[Team 5: Information Technology Initiatives]]

* [[Biological Noise and Possible Uses]]

* [[New Intro Bio Approach]]

== [[Next School Here]]==

 
 
 
----
----
 
Please see [http://meta.wikipedia.org/wiki/MediaWiki_i18n documentation on customizing the interface]
and the [http://meta.wikipedia.org/wiki/MediaWiki_User%27s_Guide User's Guide] for usage and configuration help.
 
 
[http://www.bio.davidson.edu/GCAT GCAT Main Page]

http://parts.mit.edu/igem07/index.php/Duke/Projects/bc - bacterial communication with light.

http://parts.mit.edu/igem07/index.php/Cambridge - they talk a little about making a bacterial internet, I have no idea what they mean.

http://parts.mit.edu/igem07/index.php/Tokyo_Tech - They say, “Bistability and cell-cell communication are necessary to realize our model of ‘Balanced differentiation’.”

Main Page

2013-06-19T21:20:29Z

ToEckdahl:

==[[Davidson Protocols]] 
 
==[[MWSU_protocols]] 

 
==[[Summer 2013 SynBio Project (Davidson and MWSU)]]==
 

==[[College Merit Aid]]==

 
==[[iRobot Energy Saver Project]]==

 
==[[Summer 2012 SynBio Project (Davidson and MWSU)]]==

 
==[[Synthetic Biology Network Research]]==

 
==[[Genome Assembly Project: Leland Taylor '12]]==
 

==[[Blueberry Genome Project for Bio343]]==

 

== [[Halomicrobium mukohataei Genome Fall 2009]] ==

 
== [[Halorhabdus utahensis Genome]] ==

 
==[[Network Research with Synthetic Biology]]==

 
== [[Missouri Western/Davidson SynBio 2011]] ==

 
== [[Missouri Western/Davidson iGEM2010]] ==

 
== [[Missouri Western/Davidson iGEM2009]] ==

 
== [[Davidson/Missouri Western iGEM2008]] ==

 
== [[MAGIC Tool Development]] ==

 
== [[Nova Southeastern University]] ==

 

== [[Davidson College]]== Small liberal arts college near Charlotte, NC.
A. Malcolm Campbell and Laurie J. Heyer GCAT faculty

* [http://gcat.davidson.edu/GcatWiki/index.php/User:Kahaynes Karmella A. Haynes], Visiting Assitant Professor of Biology

* [[A_Review_of_Synthetic_Biology |A Review of Synthetic Biology]] - Davidson College Synthetic Biology Seminar (Fall 2007)

* [[Laboratory Notebooks]]

* [[2009-2010 Biology Curriculum Wiki]]

* [[Team 5: Information Technology Initiatives]]

* [[Biological Noise and Possible Uses]]

* [[New Intro Bio Approach]]

== [[Next School Here]]==

 
 
 
----
----
 
Please see [http://meta.wikipedia.org/wiki/MediaWiki_i18n documentation on customizing the interface]
and the [http://meta.wikipedia.org/wiki/MediaWiki_User%27s_Guide User's Guide] for usage and configuration help.
 
 
[http://www.bio.davidson.edu/GCAT GCAT Main Page]

http://parts.mit.edu/igem07/index.php/Duke/Projects/bc - bacterial communication with light.

http://parts.mit.edu/igem07/index.php/Cambridge - they talk a little about making a bacterial internet, I have no idea what they mean.

http://parts.mit.edu/igem07/index.php/Tokyo_Tech - They say, “Bistability and cell-cell communication are necessary to realize our model of ‘Balanced differentiation’.”

Main Page

2013-06-19T21:18:30Z

ToEckdahl:

==
[[Davidson Protocols]] 
[[MWSU_protocols]] 

 
==[[Summer 2013 SynBio Project (Davidson and MWSU)]]==
 

==[[College Merit Aid]]==

 
==[[iRobot Energy Saver Project]]==

 
==[[Summer 2012 SynBio Project (Davidson and MWSU)]]==

 
==[[Synthetic Biology Network Research]]==

 
==[[Genome Assembly Project: Leland Taylor '12]]==
 

==[[Blueberry Genome Project for Bio343]]==

 

== [[Halomicrobium mukohataei Genome Fall 2009]] ==

 
== [[Halorhabdus utahensis Genome]] ==

 
==[[Network Research with Synthetic Biology]]==

 
== [[Missouri Western/Davidson SynBio 2011]] ==

 
== [[Missouri Western/Davidson iGEM2010]] ==

 
== [[Missouri Western/Davidson iGEM2009]] ==

 
== [[Davidson/Missouri Western iGEM2008]] ==

 
== [[MAGIC Tool Development]] ==

 
== [[Nova Southeastern University]] ==

 

== [[Davidson College]]== Small liberal arts college near Charlotte, NC.
A. Malcolm Campbell and Laurie J. Heyer GCAT faculty

* [http://gcat.davidson.edu/GcatWiki/index.php/User:Kahaynes Karmella A. Haynes], Visiting Assitant Professor of Biology

* [[A_Review_of_Synthetic_Biology |A Review of Synthetic Biology]] - Davidson College Synthetic Biology Seminar (Fall 2007)

* [[Laboratory Notebooks]]

* [[2009-2010 Biology Curriculum Wiki]]

* [[Team 5: Information Technology Initiatives]]

* [[Biological Noise and Possible Uses]]

* [[New Intro Bio Approach]]

== [[Next School Here]]==

 
 
 
----
----
 
Please see [http://meta.wikipedia.org/wiki/MediaWiki_i18n documentation on customizing the interface]
and the [http://meta.wikipedia.org/wiki/MediaWiki_User%27s_Guide User's Guide] for usage and configuration help.
 
 
[http://www.bio.davidson.edu/GCAT GCAT Main Page]

http://parts.mit.edu/igem07/index.php/Duke/Projects/bc - bacterial communication with light.

http://parts.mit.edu/igem07/index.php/Cambridge - they talk a little about making a bacterial internet, I have no idea what they mean.

http://parts.mit.edu/igem07/index.php/Tokyo_Tech - They say, “Bistability and cell-cell communication are necessary to realize our model of ‘Balanced differentiation’.”

Summer 2013 SynBio Project (Davidson and MWSU)

2013-05-31T15:02:11Z

ToEckdahl:

Google Hangout 11 am Wednesday (10 Central)

 

[[File:DC_MWSU_2013.jpg]] 

<center>==[[Biology]]==</center>

<center>==[[Mathematics]]==</center>

GGA results 
[[Media:DC-MWSU_GGA_results_2013.pptx]]

File:DC MWSU 2013.jpg

2013-05-31T15:01:03Z

ToEckdahl:

Summer 2013 SynBio Project (Davidson and MWSU)

2013-05-31T14:46:44Z

ToEckdahl:

Summer 2013 SynBio Project (Davidson and MWSU)

2013-05-31T14:32:00Z

ToEckdahl:

Summer 2013 SynBio Project (Davidson and MWSU)

2013-05-31T14:31:38Z

ToEckdahl:

Summer 2013 SynBio Project (Davidson and MWSU)

2013-05-31T14:30:35Z

ToEckdahl:

Google Hangout 11 am Wednesday (10 Central)

 
[[Image: DC-MWSU_2013.JPG]] 

<center>==[[Biology]]==</center>

<center>==[[Mathematics]]==</center>

GGA results 
[[Media:DC-MWSU_GGA_results_2013.pptx]]

File:DC-MWSU 2013.JPG

2013-05-31T14:30:01Z

ToEckdahl:

Summer 2013 SynBio Project (Davidson and MWSU)

2013-05-30T15:47:08Z

ToEckdahl:

Google Hangout 11 am Wednesday (10 Central)

<center>==[[Biology]]==</center>

<center>==[[Mathematics]]==</center>

GGA results 
[[Media:DC-MWSU_GGA_results_2013.pptx]]

File:DC-MWSU GGA results 2013.pptx

2013-05-30T15:45:56Z

ToEckdahl:

Talk:MWSU protocols

2013-03-06T21:28:50Z

ToEckdahl: Blanked the page

Golden Gate Assembly Protocol for BsmB1

2013-03-06T21:24:21Z

ToEckdahl: Golden Gate Assembly Protocol for BsmB1

Golden Gate Assembly Protocol for BsmB1
GGA mixture contains:
1 µL (50 ng) Plasmid
1 µL Promoter
1 µL 10X Promega Ligase Buffer
6 µL dH2O 0.5 µL BsmB1 high fidelity (HF) restriction enzyme
9.5 µL volume
Place tubes into 55°C water bath for 15 minutes. Add 0.5 µL T4 DNA Ligase to the mixture.
Turn on the PCR machine. Put tube into machine. Program it for the following cycles:
• 20 cycles
• 55°C for 10 minutes
• 37°C for 1 minute
• 16°C for 1 minute
• 22°C holding temperature
This DNA ligation is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.
Transformation of GGA BsmB1
For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.

MWSU protocols

2013-03-06T21:23:52Z

ToEckdahl: Golden Gate Assembly Protocol for BsmB1

'''Purification of DNA'''
[[Isolation of Genomic DNA from Bacteria]]

'''PCR'''

[[Standard PCR]]

[[Resuspending Oligos]]

[[Davidson_Missouri_W/colony_PCR | Colony PCR]]

'''Recombinant DNA Production'''

[[Golden Gate Assembly Protocol for BsmB1]]

[[Pouring an Agarose Gel]]

[[BioBrick Digestions for Fragment and Vector Preparation]]

[[Fragment Purification]]

[[Gibson Assembly]]

[[Direct Synthesis with Overlapping Oligos]]

[[Annealing Oligos for Cloning]]

[[Ethanol Precipitation of Vector DNA]]

[[Reducing Background from Double Digested Vector]]

'''Ligation and Transformation'''

[[BioBrick Ligations]]

[[Ligation and Transformation]]

[[Bacterial Media]]

'''Screening Clones'''

[[Diagnostic RP Digestion for Checking Insert Size]]

[[DNA Sequencing at Iowa State University]]

[[What to do with a new clone]]

'''Measuring Phenotypes'''

[[Measuring Fluorescence in Bacteria]]

MWSU protocols

2013-03-06T21:21:22Z

ToEckdahl: Golden Gate Assembly Protocol for BsmB1

'''Purification of DNA'''
[[Isolation of Genomic DNA from Bacteria]]

'''PCR'''

[[Standard PCR]]

[[Resuspending Oligos]]

[[Davidson_Missouri_W/colony_PCR | Colony PCR]]

'''Recombinant DNA Production'''

[[Golden Gate Assembly Protocol for BsmB1 ]]

[[Pouring an Agarose Gel]]

[[BioBrick Digestions for Fragment and Vector Preparation]]

[[Fragment Purification]]

[[Gibson Assembly]]

[[Direct Synthesis with Overlapping Oligos]]

[[Annealing Oligos for Cloning]]

[[Ethanol Precipitation of Vector DNA]]

[[Reducing Background from Double Digested Vector]]

'''Ligation and Transformation'''

[[BioBrick Ligations]]

[[Ligation and Transformation]]

[[Bacterial Media]]

'''Screening Clones'''

[[Diagnostic RP Digestion for Checking Insert Size]]

[[DNA Sequencing at Iowa State University]]

[[What to do with a new clone]]

'''Measuring Phenotypes'''

[[Measuring Fluorescence in Bacteria]]

Talk:MWSU protocols

2013-03-04T21:46:56Z

ToEckdahl: Golden Gate Assembly Protocol for BsmB1

Golden Gate Assembly Protocol for BsmB1

GGA mixture contains:

1 µL (50 ng) Plasmid
1 µL Promoter
1 µL 10X Promega Ligase Buffer
6 µL dH2O
0.5 µL BsmB1 high fidelity (HF) restriction enzyme
9.5 µL volume

Place tubes into 55°C water bath for 15 minutes.
Add 0.5 µL T4 DNA Ligase to the mixture.

Turn on the PCR machine. Put tube into machine.
Program it for the following cycles:
• 20 cycles
• 55°C for 10 minutes
• 37°C for 1 minute
• 16°C for 1 minute
• 22°C holding temperature

This DNA ligation is ready for transformation.
You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.

Transformation of GGA BsmB1

For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.

MWSU protocols

2013-03-04T21:44:21Z

ToEckdahl:

'''Purification of DNA'''
[[Isolation of Genomic DNA from Bacteria]]

'''PCR'''

[[Standard PCR]]

[[Resuspending Oligos]]

[[Davidson_Missouri_W/colony_PCR | Colony PCR]]

'''Recombinant DNA Production'''

[[Pouring an Agarose Gel]]

[[BioBrick Digestions for Fragment and Vector Preparation]]

[[Fragment Purification]]

[[Gibson Assembly]]

[[Direct Synthesis with Overlapping Oligos]]

[[Annealing Oligos for Cloning]]

[[Ethanol Precipitation of Vector DNA]]

[[Reducing Background from Double Digested Vector]]

'''Ligation and Transformation'''

[[BioBrick Ligations]]

[[Ligation and Transformation]]

[[Bacterial Media]]

'''Screening Clones'''

[[Diagnostic RP Digestion for Checking Insert Size]]

[[DNA Sequencing at Iowa State University]]

[[What to do with a new clone]]

'''Measuring Phenotypes'''

[[Measuring Fluorescence in Bacteria]]

MWSU protocols

2013-03-04T21:43:44Z

ToEckdahl: Golden Gate Assembly Protocol for BsmB1

'''Purification of DNA'''
Golden Gate Assembly Protocol for BsmB1

GGA mixture contains:

1 µL (50 ng) Plasmid
1 µL Promoter
1 µL 10X Promega Ligase Buffer
6 µL dH2O
0.5 µL BsmB1 high fidelity (HF) restriction enzyme
9.5 µL volume

Place tubes into 55°C water bath for 15 minutes.
Add 0.5 µL T4 DNA Ligase to the mixture.

Turn on the PCR machine. Put tube into machine.
Program it for the following cycles:
• 20 cycles
• 55°C for 10 minutes
• 37°C for 1 minute
• 16°C for 1 minute
• 22°C holding temperature

This DNA ligation is ready for transformation.
You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.

Transformation of GGA BsmB1

For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.

[[Isolation of Genomic DNA from Bacteria]]

'''PCR'''

[[Standard PCR]]

[[Resuspending Oligos]]

[[Davidson_Missouri_W/colony_PCR | Colony PCR]]

'''Recombinant DNA Production'''

[[Pouring an Agarose Gel]]

[[BioBrick Digestions for Fragment and Vector Preparation]]

[[Fragment Purification]]

[[Gibson Assembly]]

[[Direct Synthesis with Overlapping Oligos]]

[[Annealing Oligos for Cloning]]

[[Ethanol Precipitation of Vector DNA]]

[[Reducing Background from Double Digested Vector]]

'''Ligation and Transformation'''

[[BioBrick Ligations]]

[[Ligation and Transformation]]

[[Bacterial Media]]

'''Screening Clones'''

[[Diagnostic RP Digestion for Checking Insert Size]]

[[DNA Sequencing at Iowa State University]]

[[What to do with a new clone]]

'''Measuring Phenotypes'''

[[Measuring Fluorescence in Bacteria]]

Golden Gate Assembly protocol

2012-06-11T18:34:51Z

ToEckdahl:

'''Protocol to insert new promoter made from oligos into pSB1A2 with insert [http://partsregistry.org/Part:BBa_J100044 BBa_J100044]''' 

First, you need to dilute your assembled oligos. To do this, dilute them 200 fold. 
The easiest way to do this is to remove 2 µL from the 20 µL overnight mixture and place this 2 µL into a new, 1.5 mL microfuge tube. 
Now add 398 µL water to the 2 µL of cooled, annealed ologs. Mix this by stirring the tube with a yellow tip. 

Get '''two''' new, small microfuge tubes designed to fit into the PCR machine. 
One tube will be labeled "ligation", the other "plasmid only". Also put your initials on the tubes. 
1 µL diluted oligos cooled overnight (''use water instead for "plasmid only" tube'') 
9 µL ligations mixture provided by your instructor. 
use the promoter DNA for the ligation tube but use only water for the plasmid only tube. 

Ligation mixture contains: 
1 µL (10-20 ng) plasmid containing [http://partsregistry.org/Part:BBa_J100044 part J100044] 
5 µL dH2O 
1 µL 10X Promega Ligase Buffer 
1 µL 500 mM KOAc 
0.5 µL Bsa I high fidelity (HF) restriction enzyme 
0.5 µL T4 DNA Ligase 
9 µL final volume 

Turn on the PCR machine. Put '''both''' of your tubes into the machine. 
Program it for the following cylces: 
30 cycles of 37C for 1 minute/16C for 1 minute 
1 cycle of 37C for 15 minutes 
22C holding temperature 
This DNA ligation is ready for transformation. 

'''Transformations''' 
You want to do 3 transformations: 
a positive control 
your experimental ligation 
and your negative control ligation 
Use all 10 µL of the ligations for a [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html transformation]. 
You will want to perform a transformation positive control using [http://partsregistry.org/Part:BBa_K315008 K315008] which contains pLacI+RBS+RFP in plasmid pSB1A2. 

Take one tube of 100 µL competent cells and put 30 µL of cells into each of three 1.5 mL tubes labeled appropriately for each of the three [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html transformations] listed above. Plate all three transformations on LB amp plates. 

This protocols was developed at MWSU by Dr. Todd Eckdahl, and modified at Davidson College by students in Biology 111 and Romina Clemente.

Golden Gate Assembly protocol

2012-06-11T18:34:37Z

ToEckdahl:

'''Protocol to insert new promoter made from oligos into pSB1A2 with insert [http://partsregistry.org/Part:BBa_J100044 BBa_J100044]''' 

First, you need to dilute your assembled oligos. To do this, dilute them 200 fold. 
The easiest way to do this is to remove 2 µL from the 20 µL overnight mixture and place this 2 µL into a new, 1.5 mL microfuge tube. 
Now add 398 µL water to the 2 µL of cooled, annealed ologs. Mix this by stirring the tube with a yellow tip. 

Get '''two''' new, small microfuge tubes designed to fit into the PCR machine. 
One tube will be labeled "ligation", the other "plasmid only". Also put your initials on the tubes. 
1 µL diluted oligos cooled overnight (''use water instead for "plasmid only" tube'') 
9 µL ligations mixture provided by your instructor. 
use the promoter DNA for the ligation tube but use only water for the plasmid only tube. 

Ligation mixture contains: 
1 µL (10-20 ng) plasmid containing [http://partsregistry.org/Part:BBa_J100028 part J100028] 
5 µL dH2O 
1 µL 10X Promega Ligase Buffer 
1 µL 500 mM KOAc 
0.5 µL Bsa I high fidelity (HF) restriction enzyme 
0.5 µL T4 DNA Ligase 
9 µL final volume 

Turn on the PCR machine. Put '''both''' of your tubes into the machine. 
Program it for the following cylces: 
30 cycles of 37C for 1 minute/16C for 1 minute 
1 cycle of 37C for 15 minutes 
22C holding temperature 
This DNA ligation is ready for transformation. 

'''Transformations''' 
You want to do 3 transformations: 
a positive control 
your experimental ligation 
and your negative control ligation 
Use all 10 µL of the ligations for a [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html transformation]. 
You will want to perform a transformation positive control using [http://partsregistry.org/Part:BBa_K315008 K315008] which contains pLacI+RBS+RFP in plasmid pSB1A2. 

Take one tube of 100 µL competent cells and put 30 µL of cells into each of three 1.5 mL tubes labeled appropriately for each of the three [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html transformations] listed above. Plate all three transformations on LB amp plates. 

This protocols was developed at MWSU by Dr. Todd Eckdahl, and modified at Davidson College by students in Biology 111 and Romina Clemente.

Bacterial Media

2012-04-03T20:01:03Z

ToEckdahl:

Synthetic Biology Network Research

2012-03-07T13:37:16Z

ToEckdahl:

This page is designed as a community page for students at MWSU and Davidson College who are using synthetic biology to learn more about graph theory and network topology.

==Davidson College==
Our first meeting will be on Thursday, January 19, 2012. We will meet at 11 am (the common hour) in the Think Tank in the back of Belk computer lab.

'''Grading'''
* Weekly Journals (your own paper summary and those of others) = 30% final grade
* Weekly Presentations = 30% final grade
* Research Proposal by teams = 40% final grade

You must keep hard copies of your weekly journal entries in a 3-ring binder. We will grade these periodically during the semester. You will also keep copies of your papers, any drawings of ideas you have, protocols used in lab, etc.

'''Scheduling with [http://doodle.com/xkssebxsv4g3rwxk Doodle]'''

[[WEEK ONE (January 17 - 20)]]

[[WEEK TWO (January 23 - 27)]]

[[WEEK THREE (January 30 - February 3)]]

[[WEEK FOUR (February 6 - 10)]] '''WET LAB WEEK'''

[[WEEK FIVE (February 13 - 17)]]

[[WEEK SIX (February 20 - 24)]] 
Synthetic Seminar 4:30 pm Feb. 23 in '''VAC classroom''' and '''lunch''' with [http://haynes.lab.asu.edu/index.html speaker Dr. Karmella Haynes] at Arizona State University

[[WEEK SEVEN (February 27 - March 2)]]

[[WEEK EIGHT (March 5 - 9)]] '''SPRING BREAK'''

[[WEEK NINE (March 12 - 16)]]

[[WEEK TEN (March 19 - 23)]]

[[WEEK ELEVEN (March 26 - 30)]]

[[WEEK TWELVE (April 2 - 6)]]

[[WEEK THIRTEEN (April 9 - 13)]] '''INCLUDES EASTER BREAK''' 
Genomics Seminar 4:30 pm April. 11 in '''Dana 146''' and '''lunch''' with [http://guolab.uncc.edu/members/jguo4 speaker Dr. Jun-tao Gua] at UNCC Bioinformatics

[[WEEK FOURTEEN (April 16 - 20)]] 
Genomics Seminar 4:30 pm April 18 in '''Dana 146''' and '''lunch''' with [http://pediatrics.duke.edu/faculty/details/0554303 speaker Dr. Sallie Permar PhD, MD (& Davidson alumna)] at Duke University

[[WEEK FIFTEEN (April 23 - 27)]]

[[WEEK SIXTEEN (April 30 - May 4)]]

[[WEEK SEVENTEEN (May 7 - 9)]] '''READING DAY May 10'''

Over the next 14 weeks, we will read a series of papers. We have chosen some to help us get started, but as the semester progresses, you will take the lead in identifying papers. Some of these papers will be easy for you, but others will be more difficult. We will work as a group to understand what is going on. In all cases, we will use these papers to help us frame a research project that will be conducted this summer by 8 Davidson students.

We will need to become experts in the [http://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit magnetosome] produced by bacteria. We will need to identify key papers to understand what is known so far. We also need to understand what UW-Seattle iGEM2011 did with this project.

<center>'''Some possible papers'''</center>
* '''The creativity crisis'''. 
Po Bronson and Ashley Merryman 
Newsweek. July 19, 2010. page 44.

* '''Synthetic Biology Moving into the Clinic''' 
Warren C. Ruder,* Ting Lu,* James J. Collins 
Science. Vol. 333. page 1248.

* '''Engineering bacteria to solve the Burnt Pancake Problem'''. 
[http://www.jbioleng.org/content/2/1/8 Haynes, Karmella, et al.] 
Journal of Biological Engineering. Vol. 2(8): 1 – 12.

* '''Solving a Hamiltonian Path Problem with a Bacterial Computer'''. 
[http://www.jbioleng.org/content/3/1/11 Baumgardner, Jordan et al.] 
Journal of Biological Engineering. Vol. 3:11

* '''Bacterial Hash Function Using DNA-Based XOR Logic Reveals Unexpected Behavior of the LuxR Promoter'''. 
[http://www.ibc7.org/article/journal_v.php?sid=265 Brianna Pearson*, Kin H. Lau* et al.] 
Interdisciplinary Bio Central. Vol. 3, article no. 10

* '''DNA assembly for synthetic biology: from parts to pathways and beyond''' 
[http://gcat.davidson.edu/mediawiki-1.15.0/images/c/ca/Synthetic_assembly_overview.pdf Tom Ellis,*ab Tom Adieac and Geoff S. Baldwin] 
Integr. Biol., 2011, 3, 109–118

* '''Information Transduction Capacity of Noisy Biochemical Signaling Networks''' 
Raymond Cheong, Alex Rhee, Chiaochun Joanne Wang, Ilya Nemenman, Andre Levchenko 
Science. Vol. 334, page 354.

* '''Synthetic Biology: Regulating Industry Uses of New Biotechnologies''' 
Brent Erickson, Rina Singh, Paul Winters 
Science. Vol. 333, page 1254.

* '''Synthetic Biology: Integrated Gene Circuits''' 
Nagarajan Nandagopal and Michael B. Elowitz 
Science. Vol. 333, page 1244.

* '''Community Structure in Time-Dependent, Multiscale, and Multiplex Networks''' 
Peter J. Mucha, Thomas Richardson, Kevin Macon, Mason A. Porter, and Jukka-Pekka Onnela 
Science. Vol. 328. page 876-878.

* '''Stochastic Pulse Regulation in Bacterial Stress Response''' 
James C. W. Locke,* Jonathan W. Young,* Michelle Fontes, María Jesús Hernández Jiménez, Michael B. Elowitz 
Science. Vol. 334. page 366.

* '''Synthetic biology: applications come of age''' 
Ahmad S. Khalil* and James J. Collins 
Nature Review Genetics. Vol. 11. page 367.

* '''A Cultured Greigite-Producing Magnetotactic Bacterium in a Novel Group of Sulfate-Reducing Bacteria''' 
Christopher T. Lefèvre, et al. 
Science. Vol. 334. page 1720.

* '''Five hard truths for synthetic biology'''. 
Roberta Kwok 
Nature. Vol. 463. page 288.

* '''Controllability of complex networks''' 
Yang-Yu Liu1,2, Jean-Jacques Slotine3,4 & Albert-La ́szlo ́ Baraba ́si 
Nature. Vol. 473. page 167.

==MWSU==
2-17-2012

'''"Networks and confusion"'''

I have been attempting to work through the current tentative idea for our approach to network projects. I was originally confused by the idea that we would optimize an entire genome via varying RBC's, promoters, degradation tags, etc, and that we could call that a network. To make sense of the general idea of a network that we could design and eventually "train" to function, I went back to the start of what makes a network a network.

A network is a specified pathway that via interaction with a stimulus produces/degrades/builds/reacts. Antibiotic resistance is a reaction to a stimulus, but generally only uses one gene to create resistance. Whereas a metabolic pathway can use a small number to many different "nodes" or parts to the system to produce a single overall reaction in response to the stimulus.

I had an idea, this is a generalized idea, is in no way complete, but should be used as the jumping-off point. It is this: Evolution, via phenotype variation, has created the myriad of reporters and selection tools we use currently in our labs to determine if our bacteria are behaving the way we want them to. In the evolution of the various types of antibiotics we have discovered and utilize to kill bacterium, so too have those bacteria evolved to resist these killers. Looking at the scope of the interaction that a bacterium community would have to undergo in order to build a resistance to an antibiotic, one can see that it isn't likely a quick thing. however, may antibiotics are similar in function, and the resistance we encode many of our projects with are similar in their DNA construct and function as well.

'''Generalized proposal using antibiotic resistance as the stand-in for network function:'''''Italic text''

Using a bacterial strain resistant to a specific antibiotic, we add the "tools" for said bacteria to become resistant to a different but similar antibiotic by chopping up the DNA that would give them resistance and adding them to the genome of the aforementioned bacteria. Basically, we would be smashing and then giving a Rubik's cube to blind bacteria, and ordering them to put it together or we allow the new antibiotic to shoot them. We then politely poke and prod them to get to work by reassembling, chopping, and trying out new ways to use these pieces. Along the way, we use the phenotypic variation (which is inherent in all organisms) of these bacteria to train some of them (hopefully the ones that at least got part of the puzzle correct) to reach the end of a "stepwise" pathway and put the Rubik's cube back together. This pathway, keeping in mind the phenotype variation of these bacteria, doesn't have to be A-B-C-END. They might start with small a, or a triangle that strongly resembles an A, but still something similar enough to deliver the end result: resistance to a new antibiotic. This will almost assuredly be a poly-divergent pathway from start to finish. Much like many people are tall and many are small, but some of the small and the tall have dark hair or light hair, we would use the variation of our bacteria to get them from ~A through to the END. It wouldn't matter how they got to the end, even if they take shortcuts, as long as they get there, all they have to do is tell us how they did it.

Long and short, we give them the tools to solve a problem or to build a pathway, and see if they can get it right. At this stage, there are no restrictions on this idea as it is still in its infancy.

-Caleb Carr

Programming_Bacteria_for_Optimization_of_Genetic_Circuits: [[Media:Programming_Bacteria_for_Optimization_of_Genetic_Circuits.pptx‎]]

Automated Design of Synthetic RBS's to Control Protein Expression: [[Media:Automated_Design_of_Synthetic_RBS's_to_Control_Protein_Expression.pdf‎]]

Synthetic Biology Network Research

2012-03-07T13:35:03Z

ToEckdahl:

This page is designed as a community page for students at MWSU and Davidson College who are using synthetic biology to learn more about graph theory and network topology.

==Davidson College==
Our first meeting will be on Thursday, January 19, 2012. We will meet at 11 am (the common hour) in the Think Tank in the back of Belk computer lab.

'''Grading'''
* Weekly Journals (your own paper summary and those of others) = 30% final grade
* Weekly Presentations = 30% final grade
* Research Proposal by teams = 40% final grade

You must keep hard copies of your weekly journal entries in a 3-ring binder. We will grade these periodically during the semester. You will also keep copies of your papers, any drawings of ideas you have, protocols used in lab, etc.

'''Scheduling with [http://doodle.com/xkssebxsv4g3rwxk Doodle]'''

[[WEEK ONE (January 17 - 20)]]

[[WEEK TWO (January 23 - 27)]]

[[WEEK THREE (January 30 - February 3)]]

[[WEEK FOUR (February 6 - 10)]] '''WET LAB WEEK'''

[[WEEK FIVE (February 13 - 17)]]

[[WEEK SIX (February 20 - 24)]] 
Synthetic Seminar 4:30 pm Feb. 23 in '''VAC classroom''' and '''lunch''' with [http://haynes.lab.asu.edu/index.html speaker Dr. Karmella Haynes] at Arizona State University

[[WEEK SEVEN (February 27 - March 2)]]

[[WEEK EIGHT (March 5 - 9)]] '''SPRING BREAK'''

[[WEEK NINE (March 12 - 16)]]

[[WEEK TEN (March 19 - 23)]]

[[WEEK ELEVEN (March 26 - 30)]]

[[WEEK TWELVE (April 2 - 6)]]

[[WEEK THIRTEEN (April 9 - 13)]] '''INCLUDES EASTER BREAK''' 
Genomics Seminar 4:30 pm April. 11 in '''Dana 146''' and '''lunch''' with [http://guolab.uncc.edu/members/jguo4 speaker Dr. Jun-tao Gua] at UNCC Bioinformatics

[[WEEK FOURTEEN (April 16 - 20)]] 
Genomics Seminar 4:30 pm April 18 in '''Dana 146''' and '''lunch''' with [http://pediatrics.duke.edu/faculty/details/0554303 speaker Dr. Sallie Permar PhD, MD (& Davidson alumna)] at Duke University

[[WEEK FIFTEEN (April 23 - 27)]]

[[WEEK SIXTEEN (April 30 - May 4)]]

[[WEEK SEVENTEEN (May 7 - 9)]] '''READING DAY May 10'''

Over the next 14 weeks, we will read a series of papers. We have chosen some to help us get started, but as the semester progresses, you will take the lead in identifying papers. Some of these papers will be easy for you, but others will be more difficult. We will work as a group to understand what is going on. In all cases, we will use these papers to help us frame a research project that will be conducted this summer by 8 Davidson students.

We will need to become experts in the [http://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit magnetosome] produced by bacteria. We will need to identify key papers to understand what is known so far. We also need to understand what UW-Seattle iGEM2011 did with this project.

<center>'''Some possible papers'''</center>
* '''The creativity crisis'''. 
Po Bronson and Ashley Merryman 
Newsweek. July 19, 2010. page 44.

* '''Synthetic Biology Moving into the Clinic''' 
Warren C. Ruder,* Ting Lu,* James J. Collins 
Science. Vol. 333. page 1248.

* '''Engineering bacteria to solve the Burnt Pancake Problem'''. 
[http://www.jbioleng.org/content/2/1/8 Haynes, Karmella, et al.] 
Journal of Biological Engineering. Vol. 2(8): 1 – 12.

* '''Solving a Hamiltonian Path Problem with a Bacterial Computer'''. 
[http://www.jbioleng.org/content/3/1/11 Baumgardner, Jordan et al.] 
Journal of Biological Engineering. Vol. 3:11

* '''Bacterial Hash Function Using DNA-Based XOR Logic Reveals Unexpected Behavior of the LuxR Promoter'''. 
[http://www.ibc7.org/article/journal_v.php?sid=265 Brianna Pearson*, Kin H. Lau* et al.] 
Interdisciplinary Bio Central. Vol. 3, article no. 10

* '''DNA assembly for synthetic biology: from parts to pathways and beyond''' 
[http://gcat.davidson.edu/mediawiki-1.15.0/images/c/ca/Synthetic_assembly_overview.pdf Tom Ellis,*ab Tom Adieac and Geoff S. Baldwin] 
Integr. Biol., 2011, 3, 109–118

* '''Information Transduction Capacity of Noisy Biochemical Signaling Networks''' 
Raymond Cheong, Alex Rhee, Chiaochun Joanne Wang, Ilya Nemenman, Andre Levchenko 
Science. Vol. 334, page 354.

* '''Synthetic Biology: Regulating Industry Uses of New Biotechnologies''' 
Brent Erickson, Rina Singh, Paul Winters 
Science. Vol. 333, page 1254.

* '''Synthetic Biology: Integrated Gene Circuits''' 
Nagarajan Nandagopal and Michael B. Elowitz 
Science. Vol. 333, page 1244.

* '''Community Structure in Time-Dependent, Multiscale, and Multiplex Networks''' 
Peter J. Mucha, Thomas Richardson, Kevin Macon, Mason A. Porter, and Jukka-Pekka Onnela 
Science. Vol. 328. page 876-878.

* '''Stochastic Pulse Regulation in Bacterial Stress Response''' 
James C. W. Locke,* Jonathan W. Young,* Michelle Fontes, María Jesús Hernández Jiménez, Michael B. Elowitz 
Science. Vol. 334. page 366.

* '''Synthetic biology: applications come of age''' 
Ahmad S. Khalil* and James J. Collins 
Nature Review Genetics. Vol. 11. page 367.

* '''A Cultured Greigite-Producing Magnetotactic Bacterium in a Novel Group of Sulfate-Reducing Bacteria''' 
Christopher T. Lefèvre, et al. 
Science. Vol. 334. page 1720.

* '''Five hard truths for synthetic biology'''. 
Roberta Kwok 
Nature. Vol. 463. page 288.

* '''Controllability of complex networks''' 
Yang-Yu Liu1,2, Jean-Jacques Slotine3,4 & Albert-La ́szlo ́ Baraba ́si 
Nature. Vol. 473. page 167.

==MWSU==
2-17-2012

'''"Networks and confusion"'''

I have been attempting to work through the current tentative idea for our approach to network projects. I was originally confused by the idea that we would optimize an entire genome via varying RBC's, promoters, degradation tags, etc, and that we could call that a network. To make sense of the general idea of a network that we could design and eventually "train" to function, I went back to the start of what makes a network a network.

A network is a specified pathway that via interaction with a stimulus produces/degrades/builds/reacts. Antibiotic resistance is a reaction to a stimulus, but generally only uses one gene to create resistance. Whereas a metabolic pathway can use a small number to many different "nodes" or parts to the system to produce a single overall reaction in response to the stimulus.

I had an idea, this is a generalized idea, is in no way complete, but should be used as the jumping-off point. It is this: Evolution, via phenotype variation, has created the myriad of reporters and selection tools we use currently in our labs to determine if our bacteria are behaving the way we want them to. In the evolution of the various types of antibiotics we have discovered and utilize to kill bacterium, so too have those bacteria evolved to resist these killers. Looking at the scope of the interaction that a bacterium community would have to undergo in order to build a resistance to an antibiotic, one can see that it isn't likely a quick thing. however, may antibiotics are similar in function, and the resistance we encode many of our projects with are similar in their DNA construct and function as well.

'''Generalized proposal using antibiotic resistance as the stand-in for network function:'''''Italic text''

Using a bacterial strain resistant to a specific antibiotic, we add the "tools" for said bacteria to become resistant to a different but similar antibiotic by chopping up the DNA that would give them resistance and adding them to the genome of the aforementioned bacteria. Basically, we would be smashing and then giving a Rubik's cube to blind bacteria, and ordering them to put it together or we allow the new antibiotic to shoot them. We then politely poke and prod them to get to work by reassembling, chopping, and trying out new ways to use these pieces. Along the way, we use the phenotypic variation (which is inherent in all organisms) of these bacteria to train some of them (hopefully the ones that at least got part of the puzzle correct) to reach the end of a "stepwise" pathway and put the Rubik's cube back together. This pathway, keeping in mind the phenotype variation of these bacteria, doesn't have to be A-B-C-END. They might start with small a, or a triangle that strongly resembles an A, but still something similar enough to deliver the end result: resistance to a new antibiotic. This will almost assuredly be a poly-divergent pathway from start to finish. Much like many people are tall and many are small, but some of the small and the tall have dark hair or light hair, we would use the variation of our bacteria to get them from ~A through to the END. It wouldn't matter how they got to the end, even if they take shortcuts, as long as they get there, all they have to do is tell us how they did it.

Long and short, we give them the tools to solve a problem or to build a pathway, and see if they can get it right. At this stage, there are no restrictions on this idea as it is still in its infancy.

-Caleb Carr

Programming_Bacteria_for_Optimization_of_Genetic_Circuits: [[Media:Programming_Bacteria_for_Optimization_of_Genetic_Circuits.pptx‎]]

[[Media:Automated_Design_of_Synthetic_RBS's_to_Control_Protein_Expression.pdf‎ Automated Design of Synthetic RBS's to Control Protein Expression]]

File:Automated Design of Synthetic RBS's to Control Protein Expression.pdf

2012-03-07T13:33:30Z

ToEckdahl: