growth of  Methanosarcina acetivorans  with carbon monoxide as the substrate Heather Jordan Department of Biochemistry and...
A Method to the Madness…
Hypothesized Pathway 1
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Hypothesized Pathway 2
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Substrates <ul><li>Made media without any substrate </li></ul><ul><li>Used 1.5 ml inoculum per 10 ml media </li></ul><ul><...
Changing Substrates <ul><li>About 1 month required to change substrates </li></ul><ul><li>Checked cultures every 3 days </...
Scrubbing the CO  (to remove traces of O 2  and CO 2 ) <ul><li>Bubbled through solutions of  alkaline potassium pyrogallat...
 
 
Withdrawing CO for Pressurization
Pressurizing the Serum Bottle
Greatest Surface Area
Results
Nothing!!! <ul><li>Lack of turbidity </li></ul><ul><li>Media slightly yellow in color </li></ul><ul><li>Some bottles had w...
Changes in Method: <ul><li>Do not de-gas media with CO  </li></ul><ul><ul><li>Use mixed gas in glove bag </li></ul></ul><u...
While waiting for the  mixed gas tank to show up… <ul><li>Devised a new buffer system </li></ul><ul><li>K 2 HPO 4  and KH ...
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...
 
<ul><li>MA3741 MVEPLGKKVKR I AWGITGSGDQMIETYSILVDIKDRT-GVETMVFLSKEGETVMKWYHLW   </li></ul><ul><li>AF1518 MFEMEEKKKKRVAWGIT...
<ul><li>MA3741 MVEPLGKKVKR I AWGITGSGDQMIETYSILVDIKDRT-GVETMVFLSKEGETVMKWYHLW  </li></ul><ul><li>AF1518 MFEMEEKKKKRVAWGITG...
<ul><li>MA3741 M V E PLG KK V K R I AWGITG S G D QMI ET Y SILVDI K DRT-G VE TM V F L S K E G E T V MKWYHLW  </li></ul><ul>...
Acknowledgments <ul><li>Kevin Hill </li></ul>http://www.bmb.psu.edu/deptpage/ferrylab/kevin.html
Acknowledgments <ul><li>Kevin Hill </li></ul><ul><li>Francisco Cruz </li></ul>http://www.bmb.psu.edu/deptpage/ferrylab/fra...
Acknowledgments <ul><li>Kevin Hill </li></ul><ul><li>Francisco Cruz </li></ul><ul><li>Dr. Richard Ding </li></ul>http://ww...
Acknowledgments <ul><li>Kevin Hill </li></ul><ul><li>Francisco Cruz </li></ul><ul><li>Dr. Richard Ding </li></ul><ul><li>D...
And Special Thanks To: <ul><li>Sarah Lawrence </li></ul>http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html
And Special Thanks To: <ul><li>Sarah Lawrence </li></ul>http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html <ul><li>Dr. ...
And Special Thanks To: <ul><li>Sarah Lawrence </li></ul>http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html <ul><li>Dr. ...
And Special Thanks To: <ul><li>Sarah Lawrence </li></ul><ul><li>Dr. Andrea Gorrell </li></ul><ul><li>Eric Patridge </li></...
And who could forget… <ul><li>Christie Brosius </li></ul>http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html http://www....
 
<ul><li>Birren, B. et al., “The Genome of  Methanosarcina acetivorans  reveals unprecedented metabolic and physiological d...
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Growth Of Methanosarcina Acetivorans On Carbon Monoxide Substrate

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Metabolic study of M. acetivorans with CO as the sole carbon source.

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Growth Of Methanosarcina Acetivorans On Carbon Monoxide Substrate

  1. 3. 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
  2. 4. A Method to the Madness…
  3. 5. Hypothesized Pathway 1
  4. 26. Hypothesized Pathway 2
  5. 41. Substrates <ul><li>Made media without any substrate </li></ul><ul><li>Used 1.5 ml inoculum per 10 ml media </li></ul><ul><li>Inoculated with both TMA and acetate-grown cultures of strain C 2 A </li></ul>
  6. 42. Changing Substrates <ul><li>About 1 month required to change substrates </li></ul><ul><li>Checked cultures every 3 days </li></ul><ul><li>Labeled CO cultures with inoculum substrate </li></ul><ul><li>Since TMA is the favorite food, we expected that this substrate would be difficult to wean from ( should grow better from acetate culture ) </li></ul>
  7. 43. Scrubbing the CO (to remove traces of O 2 and CO 2 ) <ul><li>Bubbled through solutions of alkaline potassium pyrogallate and NaOH connected in series. </li></ul>http://www.calibredigital.com/Services/index.html
  8. 46. Withdrawing CO for Pressurization
  9. 47. Pressurizing the Serum Bottle
  10. 48. Greatest Surface Area
  11. 49. Results
  12. 50. Nothing!!! <ul><li>Lack of turbidity </li></ul><ul><li>Media slightly yellow in color </li></ul><ul><li>Some bottles had white precipitate in them </li></ul><ul><li>Open them up to see if they turn pink </li></ul><ul><li>Re-make the media and start over </li></ul>Failure
  13. 51. Changes in Method: <ul><li>Do not de-gas media with CO </li></ul><ul><ul><li>Use mixed gas in glove bag </li></ul></ul><ul><li>Do not pressurize bottles with pure CO </li></ul><ul><ul><li>Use CO:CO 2 mix (80:20) </li></ul></ul><ul><li>Do not add inoculum to bottles without any substrate </li></ul><ul><ul><li>Add 20 % the usual amount of TMA or methanol and don’t bother trying acetate </li></ul></ul>We shall wait and see what happens…
  14. 52. While waiting for the mixed gas tank to show up… <ul><li>Devised a new buffer system </li></ul><ul><li>K 2 HPO 4 and KH 2 PO 4 </li></ul><ul><li>Leave out NaHCO 3 </li></ul><ul><li>Gas with pure CO </li></ul><ul><li>Add 20% TMA to media as planned </li></ul><ul><li>And the result of all this? </li></ul>
  15. 53. To be continued…
  16. 54. 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
  17. 56. <ul><li>MA3741 MVEPLGKKVKR I AWGITGSGDQMIETYSILVDIKDRT-GVETMVFLSKEGETVMKWYHLW </li></ul><ul><li>AF1518 MFEMEEKKKKRVAWGITGSGDRLPETVDVMLEVKKEFPDVEIAVYLSKAGAQVVKYYKLM </li></ul><ul><li>MA0696 ------MSFKS I AWGITGAGHFLDRSYQVFKELKQRDPELSVNTYISRAAEEVLRMYGLE </li></ul><ul><li>MA3741 </li></ul><ul><li>AF1518 </li></ul><ul><li>MA0696 </li></ul><ul><li>MA3741 </li></ul><ul><li>AF1518 </li></ul><ul><li>MA0696 </li></ul><ul><li>MA3741 </li></ul><ul><li>AF1518 </li></ul><ul><li>MA0696 NGVTDQIELLKCKGCGICKELCPYNAIKGICKEVLVRDVDMRNVEIVKGLQGITVLESPE </li></ul><ul><li>MA3741 DIYDIFGLEKPSEDITIKVKERKKSKTRK </li></ul><ul><li>AF1518 DIPKVF---------------------RKHFGKS---- </li></ul><ul><li>MA0696 AILELF----------------------- </li></ul>
  18. 57. <ul><li>MA3741 MVEPLGKKVKR I AWGITGSGDQMIETYSILVDIKDRT-GVETMVFLSKEGETVMKWYHLW </li></ul><ul><li>AF1518 MFEMEEKKKKRVAWGITGSGDRLPETVDVMLEVKKEFPDVEIAVYLSKAGAQVVKYYKLM </li></ul><ul><li>MA0696 ------MSFKS I AWGITGAGHFLDRSYQVFKELKQRDPELSVNTYISRAAEEVLRMYGLE </li></ul><ul><li>MA3741 DKIQKDFP------------NFKVDAGPNSPFIAGPLQMGYYDFLLIAPATANTVAKIVYGIADTL </li></ul><ul><li>AF1518 DTLKENFG-----------AVRVEIDANTPFLAGQIQVGRYEFLLIAPATSNTVAKIAMGIADSL </li></ul><ul><li>MA0696 QKLVKISGGDYLEEIFRESEQGSSSPKVGRFLLDRYDALFVTPATSNTVSKIAYGIADSL </li></ul><ul><li>MA3741 VTNAVSQTAKGKTPIFILPVDQKRGTVKTAAPS--------------------------- </li></ul><ul><li>AF1518 LANAAIMGLKAYVPLYIMPSDYKEGEVITKLPD--------------------------- </li></ul><ul><li>MA0696 VTNAVAQAVKGKVPVYVVPVD-IEGSIISEMPYNIDRKQCRHCETCPPRENCPHGAITEK </li></ul><ul><li>MA3741 ----------------------------------------------------GKAFELSMREVDVTNSEKLAQMENISVLASPY </li></ul><ul><li>AF1518 -----------------------------------------------------GRDLRLRVRKEDVEHVRKLAQMDGVTILEKPE </li></ul><ul><li>MA0696 NGVTDQIELLKCKGCGICKELCPYNAIKGICKEVLVRDVDMRNVEIVKGLQGITVLESPE </li></ul><ul><li>MA3741 DIYDIFGLEKPSEDITIKVKERKKSKTRK </li></ul><ul><li>AF1518 DIPKVF---------------------RKHFGKS---- </li></ul><ul><li>MA0696 AILELF----------------------- </li></ul>
  19. 58. <ul><li>MA3741 M V E PLG KK V K R I AWGITG S G D QMI ET Y SILVDI K DRT-G VE TM V F L S K E G E T V MKWYHLW </li></ul><ul><li>AF1518 M F E MEE KK K K R V AWGITG S G D R L P ET VD V ML E V K KEF P D VE IA V Y L S K A G A Q V VKYYKLM </li></ul><ul><li>MA0696 ------MSF K S I AWGITG A G HF L DRS Y Q V FK E L K QRD P ELSVNT Y I S R A A E E V LRMYGLE </li></ul><ul><li>MA3741 DKIQ K D F P------------N F K V DAGP N S PF I A G PL Q M G Y Y D F L LIA PAT A NTV A KI V Y GIAD T L </li></ul><ul><li>AF1518 DT L KEN F G -----------AV R V EIDA N T PF L A G QI Q V G R Y E F L LIA PAT S NTV A KI A M GIAD S L </li></ul><ul><li>MA0696 QK L V K IS G GDYLEEI F R ESEQG S SSPKV G RFLLD R Y D A L FVT PAT S NTV S KI A Y GIAD S L </li></ul><ul><li>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--------------------------- </li></ul><ul><li>AF1518 LA NA AIMGLKAY V P L Y I M P S D Y K E G E V I T KL P D--------------------------- </li></ul><ul><li>MA0696 VT NA V A Q AVKGK V P V Y VV P V D -I E G SI I SEM P YNIDRKQCRHCETCPPRENCPHGAITEK </li></ul><ul><li>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 </li></ul><ul><li>AF1518 ----------------------------------------------------- G RDLR L R V R KE D V EH V R KLAQM D G V T I L E K P E </li></ul><ul><li>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 </li></ul><ul><li>MA3741 D I YDI F GLEKPSEDITIKV K ERK KS KTRK </li></ul><ul><li>AF1518 D I PKV F ---------------------R K HFG KS ---- </li></ul><ul><li>MA0696 A I LEL F ----------------------- </li></ul>
  20. 59. Acknowledgments <ul><li>Kevin Hill </li></ul>http://www.bmb.psu.edu/deptpage/ferrylab/kevin.html
  21. 60. Acknowledgments <ul><li>Kevin Hill </li></ul><ul><li>Francisco Cruz </li></ul>http://www.bmb.psu.edu/deptpage/ferrylab/frank.html
  22. 61. Acknowledgments <ul><li>Kevin Hill </li></ul><ul><li>Francisco Cruz </li></ul><ul><li>Dr. Richard Ding </li></ul>http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html
  23. 62. Acknowledgments <ul><li>Kevin Hill </li></ul><ul><li>Francisco Cruz </li></ul><ul><li>Dr. Richard Ding </li></ul><ul><li>Dr. J.G. Ferry </li></ul>http://www.bmb.psu.edu/deptpage/ferry.htm
  24. 63. And Special Thanks To: <ul><li>Sarah Lawrence </li></ul>http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html
  25. 64. And Special Thanks To: <ul><li>Sarah Lawrence </li></ul>http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html <ul><li>Dr. Andrea Gorrell </li></ul>
  26. 65. And Special Thanks To: <ul><li>Sarah Lawrence </li></ul>http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html <ul><li>Dr. Andrea Gorrell </li></ul><ul><li>Eric Patridge </li></ul>
  27. 66. And Special Thanks To: <ul><li>Sarah Lawrence </li></ul><ul><li>Dr. Andrea Gorrell </li></ul><ul><li>Eric Patridge </li></ul><ul><li>Sabrina Zimmerman </li></ul>Sorry, no photo available (Cameron lab does not have a website).
  28. 67. And who could forget… <ul><li>Christie Brosius </li></ul>http://www.bmb.psu.edu/deptpage/ferrylab/lablist.html http://www.atpictures.com/display.php?path=/ben&loc=serva8&img=ba95.jpg
  29. 69. <ul><li>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 . </li></ul>The End 5  m

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