IGEM:MIT/2006/Notebook/2006-8-11: Difference between revisions

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==To do==
==To do==
#smell overnight IK smell testers and '''after making glycerols from regular batch''' then add precursor to the daytime smell testers
#Recontact Jose Aleman in Stephanopoulos lab. I (austin) talked to him today. He said they fixed their GC yesterday.
#analyze sequencing from weds [full assemblies]
#smell overnight IK smell testers and '''after making glycerols from regular batch''' then add precursor to the daytime smell testers '''-done'''
#analyze sequencing from weds [full assemblies] '''veena and kate, afternoon'''
#List Biobrick Molecules
#List Biobrick Molecules
#add pchBA to registry
#*Lets update our index with our numbering system and all the biobrick constructs we believe that we have (and then we can check them off as we get the sequence results back. Also, '''BO''' could you put in links to each part's registry page on the index!)
#*Lets update our index with our numbering system and all the biobrick constructs we believe that we have (and then we can check them off as we get the sequence results back. Also, '''BO''' could you put in links to each part's registry page on the index!)
#digest WGD with EP and run on a gel with 8/10 pUCP22:EP digest
#digest WGD with EP and run on a gel with 8/10 pUCP22:EP digest '''digest started 12:30'''
#ligate WGD into EP cut pUCP22 shuttle vector and transform into Top10 in the pm
#ligate WGD into EP cut pUCP22 shuttle vector and transform into Top10 in the pm
#autoclave waste
#autoclave waste
#digest pCHBA with E and S and backbone with E and S and ligate
#digest pCHBA with E and S and backbone with E and S and ligate '''digest started 12:30'''
#mutagenesis of BAT2, transformation of BAT2, ATF1, and THI3 mutagenesis
#mutagenesis of BAT2, transformation of BAT2, ATF1, and THI3 mutagenesis '''Bo and Stephen pm'''
#LCs of R0040.E0840 and osmY.E0840s
#LCs of R0040.E0840 and osmY.E0840s
#GC of smell tests
#GC of smell tests '''decided to run it when we have more controlled samples to run'''
#miniprep RBS.coding.terminators to inverters
#miniprep RBS.coding.terminators to inverters '''digests of these started at 12:30'''


==Preliminary Smell Test: Indole KO Strain (AG)==
==''Preliminary'' Smell Test I ([[User:Dagreen|André]])==


===Experiment description===
===Experiment description===


*We would like to know the amount of time it takes to produce the final volatile compound.  We added SA (2mM) and IA-OH (10mM) to 5mL pre-grown Indole KO cultures.   
*We would like to know the amount of time it takes to produce the final volatile compound.   
**I added SA (2mM) and IA-OH (10mM) to 5mL pre-grown Indole KO cultures.   
**Cultures containing J45100 and J45220 were further incubated at room temperature (25C)
**Cultures containing J45120, J45170, and J45200 were further incubated (with vigorous shaking) at 37C
**'''After 240 min (4hrs), I continued the experiment with just the J45200 in order to determine the amount of time it took the cells to stop producing the scent (either because the cells have reached stationary phase, started dying, etc.)'''


{| border="1" cellpadding="5"
{| border="1" cellpadding="5"
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|'''smells pretty bad, i.e. like regular Indole KO cells'''
|'''smells pretty bad, i.e. like regular Indole KO cells'''
|'''smells pretty bad, i.e. like regular Indole KO cells'''
|'''smells pretty bad, i.e. like regular Indole KO cells'''
|'''faint smell of banana; smell of precursor lingering'''
|'''faint smell of banana; smell of IA-OH lingering'''
|''isoamyl alcohol greatly overpowering''
|''IA-OH greatly overpowering''
|-
|-
|'''30 min'''
|'''30 min'''
Line 39: Line 45:
|'''smells pretty bad, i.e. like regular Indole KO cells'''
|'''smells pretty bad, i.e. like regular Indole KO cells'''
|'''noticeable smell of banana, stronger than at 15 min'''
|'''noticeable smell of banana, stronger than at 15 min'''
|''isoamyl alcohol greatly overpowering''
|''IA-OH greatly overpowering''
|-
|-
|'''60 min'''
|'''60 min'''
|''slight hint of mint; normal IKO-smell prevalent''
|'''noticeable mint smell; normal IKO-smell prevalent'''
|'''smells pretty bad, i.e. like regular Indole KO cells'''
|'''powerful banana scent'''
|''IA-OH greatly overpowering''
|-
|-
|'''90 min'''
|'''90 min'''
|''slight hint of mint; normal IKO-smell prevalent''
|'''noticeable mint smell; normal IKO-smell prevalent'''
|'''smells pretty bad, i.e. like regular Indole KO cells'''
|'''strong banana scent'''
|''IA-OH greatly overpowering''
|-
|-
|'''120 min'''
|'''120 min'''
|''slight hint of mint; normal IKO-smell prevalent''
|'''noticeable mint smell; normal IKO-smell prevalent'''
|'''smells pretty bad, i.e. like regular Indole KO cells'''
|'''strong banana scent'''
|''banana smell is faint; IA-OH smell prevalent''
|-
|-
|'''150 min'''
|'''240 min'''
|''very slight hint of mint; normal IKO-smell prevalent''
|'''faint mint smell; normal IKO-smell prevalent'''
|'''smells pretty bad, i.e. like regular Indole KO cells'''
|'''weakened banana scent'''
|''IA-OH smell prevalent''
|}
|}
====Conclusions====
*osmY is probably not correctly assembled in the BioBrick (J45140).
*Some smell is obtained from cells that are incubated at 25C, indicating that ATF1 protein is present in a sufficient amount after overnight culturing.
*Smell is obtained much more quickly when incubation is resumed at 37C as opposed to 25C.  This is likely because the ATF1 protein present in the overnight culture is supplemented by increased protein production at 37C.
*Because smell (banana) was detected after only 15 min of incubation at 37C (J45200), this means that sufficient IA-OH enters the cells within 15 minutes of addition.
==ligations completed (Kate and Veena)==
#pUCP22 [EP] + J45100 [EP]  ->amp/gent
#pUCP22 [EP] + J45120 [EP]  ->amp/gent
#pchBA [ES] + pSB1AK3 [ES]  ->amp/kan
#pchBA [ES] + pSB1A3-1 [ES] ->amp
#Q04400 [ES] + J45099 [XP] + pSB1AT3 [EP] ->amp/tet
#Q04400 [ES] + J45119 [XP] + pSB1AT3 [EP] ->amp/tet
#Q04400 [ES] + J45199 [XP] + pSB1AT3 [EP] ->amp/tet
#Q04400 [ES] + J45219 [XP] + pSB1AT3 [EP] ->amp/tet
#osmY annealed [ES] + E0840 [XP] + pSB1AT3 [EP] ->amp/tet

Latest revision as of 16:02, 11 August 2006

To do

  1. Recontact Jose Aleman in Stephanopoulos lab. I (austin) talked to him today. He said they fixed their GC yesterday.
  2. smell overnight IK smell testers and after making glycerols from regular batch then add precursor to the daytime smell testers -done
  3. analyze sequencing from weds [full assemblies] veena and kate, afternoon
  4. List Biobrick Molecules
  5. add pchBA to registry
    • Lets update our index with our numbering system and all the biobrick constructs we believe that we have (and then we can check them off as we get the sequence results back. Also, BO could you put in links to each part's registry page on the index!)
  6. digest WGD with EP and run on a gel with 8/10 pUCP22:EP digest digest started 12:30
  7. ligate WGD into EP cut pUCP22 shuttle vector and transform into Top10 in the pm
  8. autoclave waste
  9. digest pCHBA with E and S and backbone with E and S and ligate digest started 12:30
  10. mutagenesis of BAT2, transformation of BAT2, ATF1, and THI3 mutagenesis Bo and Stephen pm
  11. LCs of R0040.E0840 and osmY.E0840s
  12. GC of smell tests decided to run it when we have more controlled samples to run
  13. miniprep RBS.coding.terminators to inverters digests of these started at 12:30

Preliminary Smell Test I (André)

Experiment description

  • We would like to know the amount of time it takes to produce the final volatile compound.
    • I added SA (2mM) and IA-OH (10mM) to 5mL pre-grown Indole KO cultures.
    • Cultures containing J45100 and J45220 were further incubated at room temperature (25C)
    • Cultures containing J45120, J45170, and J45200 were further incubated (with vigorous shaking) at 37C
    • After 240 min (4hrs), I continued the experiment with just the J45200 in order to determine the amount of time it took the cells to stop producing the scent (either because the cells have reached stationary phase, started dying, etc.)
J45100 J45120 J45170 J45200 J45220
15 min smells pretty bad, i.e. like regular Indole KO cells smells pretty bad, i.e. like regular Indole KO cells smells pretty bad, i.e. like regular Indole KO cells faint smell of banana; smell of IA-OH lingering IA-OH greatly overpowering
30 min slight hint of mint; normal IKO-smell prevalent very slight hint of mint; normal IKO-smell prevalent smells pretty bad, i.e. like regular Indole KO cells noticeable smell of banana, stronger than at 15 min IA-OH greatly overpowering
60 min slight hint of mint; normal IKO-smell prevalent noticeable mint smell; normal IKO-smell prevalent smells pretty bad, i.e. like regular Indole KO cells powerful banana scent IA-OH greatly overpowering
90 min slight hint of mint; normal IKO-smell prevalent noticeable mint smell; normal IKO-smell prevalent smells pretty bad, i.e. like regular Indole KO cells strong banana scent IA-OH greatly overpowering
120 min slight hint of mint; normal IKO-smell prevalent noticeable mint smell; normal IKO-smell prevalent smells pretty bad, i.e. like regular Indole KO cells strong banana scent banana smell is faint; IA-OH smell prevalent
240 min very slight hint of mint; normal IKO-smell prevalent faint mint smell; normal IKO-smell prevalent smells pretty bad, i.e. like regular Indole KO cells weakened banana scent IA-OH smell prevalent

Conclusions

  • osmY is probably not correctly assembled in the BioBrick (J45140).
  • Some smell is obtained from cells that are incubated at 25C, indicating that ATF1 protein is present in a sufficient amount after overnight culturing.
  • Smell is obtained much more quickly when incubation is resumed at 37C as opposed to 25C. This is likely because the ATF1 protein present in the overnight culture is supplemented by increased protein production at 37C.
  • Because smell (banana) was detected after only 15 min of incubation at 37C (J45200), this means that sufficient IA-OH enters the cells within 15 minutes of addition.

ligations completed (Kate and Veena)

  1. pUCP22 [EP] + J45100 [EP] ->amp/gent
  2. pUCP22 [EP] + J45120 [EP] ->amp/gent
  3. pchBA [ES] + pSB1AK3 [ES] ->amp/kan
  4. pchBA [ES] + pSB1A3-1 [ES] ->amp
  5. Q04400 [ES] + J45099 [XP] + pSB1AT3 [EP] ->amp/tet
  6. Q04400 [ES] + J45119 [XP] + pSB1AT3 [EP] ->amp/tet
  7. Q04400 [ES] + J45199 [XP] + pSB1AT3 [EP] ->amp/tet
  8. Q04400 [ES] + J45219 [XP] + pSB1AT3 [EP] ->amp/tet
  9. osmY annealed [ES] + E0840 [XP] + pSB1AT3 [EP] ->amp/tet
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