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Growth Of Methanosarcina Acetivorans On Carbon Monoxide Substrate

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Information about Growth Of Methanosarcina Acetivorans On Carbon Monoxide Substrate

Published on June 1, 2008

Author: Water_Xta1

Source: slideshare.net

Description

Metabolic study of M. acetivorans with CO as the sole carbon source.
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growth of Methanosarcina acetivorans with carbon monoxide as the substrate Heather Jordan Department of Biochemistry and Molecular Biology The 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 C 2 A

Made media without any substrate

Used 1.5 ml inoculum per 10 ml media

Inoculated with both TMA and acetate-grown cultures of strain C 2 A

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 )

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 O 2 and CO 2 ) Bubbled through solutions of alkaline potassium pyrogallate and NaOH connected in series. http://www.calibredigital.com/Services/index.html

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

 

 

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 Failure

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: Do not de-gas media with CO Use mixed gas in glove bag Do not pressurize bottles with pure CO Use CO:CO 2 mix (80:20) 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…

Do not de-gas media with CO

Use mixed gas in glove bag

Do not pressurize bottles with pure CO

Use CO:CO 2 mix (80:20)

Do not add inoculum to bottles without any substrate

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

While waiting for the mixed gas tank to show up… Devised a new buffer system K 2 HPO 4 and KH 2 PO 4 Leave out NaHCO 3 Gas with pure CO Add 20% TMA to media as planned And the result of all this?

Devised a new buffer system

K 2 HPO 4 and KH 2 PO 4

Leave out NaHCO 3

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 To A Lab Near You

 

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MA3741 MVEPLGKKVKR I AWGITGSGDQMIETYSILVDIKDRT-GVETMVFLSKEGETVMKWYHLW

AF1518 MFEMEEKKKKRVAWGITGSGDRLPETVDVMLEVKKEFPDVEIAVYLSKAGAQVVKYYKLM

MA0696 ------MSFKS I AWGITGAGHFLDRSYQVFKELKQRDPELSVNTYISRAAEEVLRMYGLE

MA3741

AF1518

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MA0696

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MA0696 NGVTDQIELLKCKGCGICKELCPYNAIKGICKEVLVRDVDMRNVEIVKGLQGITVLESPE

MA3741 DIYDIFGLEKPSEDITIKVKERKKSKTRK

AF1518 DIPKVF---------------------RKHFGKS----

MA0696 AILELF-----------------------

MA3741 MVEPLGKKVKR I AWGITGSGDQMIETYSILVDIKDRT-GVETMVFLSKEGETVMKWYHLW AF1518 MFEMEEKKKKRVAWGITGSGDRLPETVDVMLEVKKEFPDVEIAVYLSKAGAQVVKYYKLM MA0696 ------MSFKS I AWGITGAGHFLDRSYQVFKELKQRDPELSVNTYISRAAEEVLRMYGLE MA3741 DKIQKDFP------------NFKVDAGPNSPFIAGPLQMGYYDFLLIAPATANTVAKIVYGIADTL AF1518 DTLKENFG-----------AVRVEIDANTPFLAGQIQVGRYEFLLIAPATSNTVAKIAMGIADSL MA0696 QKLVKISGGDYLEEIFRESEQGSSSPKVGRFLLDRYDALFVTPATSNTVSKIAYGIADSL MA3741 VTNAVSQTAKGKTPIFILPVDQKRGTVKTAAPS--------------------------- AF1518 LANAAIMGLKAYVPLYIMPSDYKEGEVITKLPD--------------------------- MA0696 VTNAVAQAVKGKVPVYVVPVD-IEGSIISEMPYNIDRKQCRHCETCPPRENCPHGAITEK MA3741 ----------------------------------------------------GKAFELSMREVDVTNSEKLAQMENISVLASPY AF1518 -----------------------------------------------------GRDLRLRVRKEDVEHVRKLAQMDGVTILEKPE MA0696 NGVTDQIELLKCKGCGICKELCPYNAIKGICKEVLVRDVDMRNVEIVKGLQGITVLESPE MA3741 DIYDIFGLEKPSEDITIKVKERKKSKTRK AF1518 DIPKVF---------------------RKHFGKS---- MA0696 AILELF-----------------------

MA3741 MVEPLGKKVKR I AWGITGSGDQMIETYSILVDIKDRT-GVETMVFLSKEGETVMKWYHLW

AF1518 MFEMEEKKKKRVAWGITGSGDRLPETVDVMLEVKKEFPDVEIAVYLSKAGAQVVKYYKLM

MA0696 ------MSFKS I AWGITGAGHFLDRSYQVFKELKQRDPELSVNTYISRAAEEVLRMYGLE

MA3741 DKIQKDFP------------NFKVDAGPNSPFIAGPLQMGYYDFLLIAPATANTVAKIVYGIADTL

AF1518 DTLKENFG-----------AVRVEIDANTPFLAGQIQVGRYEFLLIAPATSNTVAKIAMGIADSL

MA0696 QKLVKISGGDYLEEIFRESEQGSSSPKVGRFLLDRYDALFVTPATSNTVSKIAYGIADSL

MA3741 VTNAVSQTAKGKTPIFILPVDQKRGTVKTAAPS---------------------------

AF1518 LANAAIMGLKAYVPLYIMPSDYKEGEVITKLPD---------------------------

MA0696 VTNAVAQAVKGKVPVYVVPVD-IEGSIISEMPYNIDRKQCRHCETCPPRENCPHGAITEK

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MA0696 NGVTDQIELLKCKGCGICKELCPYNAIKGICKEVLVRDVDMRNVEIVKGLQGITVLESPE

MA3741 DIYDIFGLEKPSEDITIKVKERKKSKTRK

AF1518 DIPKVF---------------------RKHFGKS----

MA0696 AILELF-----------------------

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MA3741 VT NA V S Q TAKGKT P IF I L P V D Q K R G T V K T AA P S---------------------------

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MA3741 ---------------------------------------------------- G KAF E L SM R E V D V T N S E KLAQM EN I S V L A S P Y

AF1518 ----------------------------------------------------- G RDLR L R V R KE D V EH V R KLAQM D G V T I L E K P E

MA0696 NGVTDQIELLKCKGCGICKELCPYNAIK G ICK E VL V R D V D MR N V E IVKGLQ G I T V L E S P E

MA3741 D I YDI F GLEKPSEDITIKV K ERK KS KTRK

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MA0696 A I LEL F -----------------------

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

Kevin Hill

Acknowledgments Kevin Hill Francisco Cruz http://www.bmb.psu.edu/deptpage/ferrylab/frank.html

Kevin Hill

Francisco Cruz

Acknowledgments Kevin Hill Francisco Cruz Dr. Richard Ding http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html

Kevin Hill

Francisco Cruz

Dr. Richard Ding

Acknowledgments Kevin Hill Francisco Cruz Dr. Richard Ding Dr. J.G. Ferry http://www.bmb.psu.edu/deptpage/ferry.htm

Kevin Hill

Francisco Cruz

Dr. Richard Ding

Dr. J.G. Ferry

And Special Thanks To: Sarah Lawrence http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html

Sarah Lawrence

And Special Thanks To: Sarah Lawrence http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html Dr. Andrea Gorrell

Sarah Lawrence

Dr. Andrea Gorrell

And Special Thanks To: Sarah Lawrence http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html Dr. Andrea Gorrell Eric Patridge

Sarah Lawrence

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).

Sarah Lawrence

Dr. Andrea Gorrell

Eric Patridge

Sabrina Zimmerman

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

Christie Brosius

 

Birren, B. et al., “The Genome of Methanosarcina acetivorans reveals unprecedented metabolic and physiological diversity,” http://www.genome.org/cgi/doi/10.1101/gr.223902 . The End 5  m

Birren, B. et al., “The Genome of Methanosarcina acetivorans reveals unprecedented metabolic and physiological diversity,” http://www.genome.org/cgi/doi/10.1101/gr.223902 .

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