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This topic has been modified from its original version. It has been formatted

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growth of Methanosarcina acetivorans with carbon

monoxide as the substrate

Heather Jordan

Department of Biochemistry and Molecular BiologyThe Pennsylvania State University

University Park, Pennsylvania 16802

A Method to the Madness…

Hypothesized Pathway 1

Hypothesized Pathway 2

Substrates• Made media

without any substrate

• Used 1.5 ml inoculum per 10 ml media

• Inoculated with both TMA and acetate-grown cultures of strain C2A

Changing Substrates

• About 1 month required to change substrates

• Checked cultures every 3 days

• Labeled CO cultures with inoculum substrate

• Since TMA is the favorite food, we expected that this substrate would be difficult to wean from (should grow better from acetate culture)

Scrubbing the CO (to remove traces of O2 and CO2)

• Bubbled through solutions of alkaline potassium pyrogallate and NaOH connected in series.

http://www.calibredigital.com/Services/index.html

Withdrawing CO for Pressurization

Pressurizing the Serum Bottle

Greatest Surface Area

Results

Nothing!!!

• Lack of turbidity

• Media slightly yellow in color

• Some bottles had white precipitate in them

• Open them up to see if they turn pink

• Re-make the media and start over

Changes in Method:1) Do not de-gas media with CO

Use mixed gas in glove bag

1) Do not pressurize bottles with pure CO Use CO:CO2 mix (80:20)

2) Do not add inoculum to bottles without any substrate

Add 20 % the usual amount of TMA or methanol and don’t bother trying acetate

We shall wait and see what happens…

While waiting for the mixed gas tank to show up…

• Devised a new buffer system

• K2HPO4 and KH2PO4

• Leave out NaHCO3

• Gas with pure CO

• Add 20% TMA to media as planned

• And the result of all this?

To be continued…

Creating ISF Knockouts!

Boxing template from: http://joyjobs.com/esl/tefl_1.htm

Kevin’s head from:http://www.bmb.psu.edu/debtpage/ferrylab/evenmorepicturesa.html

Coming Soon Coming Soon To A Lab Near YouTo A Lab Near You

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MA3741VTNAVSQTAKGKTPIFILPVDQKRGTVKTAAPS---------------------------

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Acknowledgments

• Kevin Hill

http://www.bmb.psu.edu/deptpage/ferrylab/kevin.html

Acknowledgments

• Kevin Hill

• Francisco Cruz

http://www.bmb.psu.edu/deptpage/ferrylab/frank.html

Acknowledgments

• Kevin Hill

• Francisco Cruz

• Dr. Richard Ding

http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html

Acknowledgments

• Kevin Hill

• Francisco Cruz

• Dr. Richard Ding

• Dr. J.G. Ferry

http://www.bmb.psu.edu/deptpage/ferry.htm

And Special Thanks To:

• Sarah Lawrence

http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html

And Special Thanks To:

• Sarah Lawrence

http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html

• Dr. Andrea Gorrell

And Special Thanks To:

• Sarah Lawrence

http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html

• Dr. Andrea Gorrell

• Eric Patridge

And Special Thanks To:

• Sarah Lawrence

• Dr. Andrea Gorrell

• Eric Patridge

• Sabrina Zimmerman

Sorry, no photo available (Cameron lab does not have a website).

And who could forget…

• Christie Brosius

http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html

http://www.atpictures.com/display.php?path=/ben&loc=serva8&img=ba95.jpg

5 m5 mBirren, B. et al., “The Genome of Birren, B. et al., “The Genome of Methanosarcina acetivoransMethanosarcina acetivorans reveals unprecedented reveals unprecedented

metabolic and physiological diversity,” metabolic and physiological diversity,” http://www.genome.org/cgi/doi/10.1101/gr.223902http://www.genome.org/cgi/doi/10.1101/gr.223902..