IGEM:MIT/2006/Notebook/2006-7-13

Exciting Overnight Results

1. Our SA biosynthesis cells grew up successfully from stab
2. Indole-knockout cells A, B, and C grew successfully.... and don't smell like indole. They just smell like LB!!!
   • Come smell!!
   • Also, even though strain YYC912 seems to be working well for us, I decided to order 2 other strains of tnaA mutants to test. They should arrive shortly.

To do

1. Repeat Ligation of coding region to terminator
   • We had no transformants. However, gel indicates that digest worked. We have left-over digest, so will use that and try ligation and transformation again.
2. Sequencing results are in. We need to compare with Genbank sequences
3. Repeat osmY promoter PCR from E. coli with shorter annealing time
   • PCR clean-up
   • run a gel to check
4. GC work on methyl salicylate (Veena am)
5. Clean out fridge trays
6. Experiment with new SA biosynthesis cells and indole-negative cells
   • Miniprep SA cells
   • Transform SA plasmid (pUC18, Amp) and SAMT (pET28, Kan) into Top10 cells together
     • check that Oris are different and compatible for the two plasmids
   • Make YYC912 indole-knockout cells chemically competent
     • someone needs to read protocols, set up overnight cultures, and be ready for a 3 hr process tomorrow
7. Digest BAMT mutagenesis
8. Informal meeting at 4 to discuss progress/plan

Mutagenesis Check

We need to check and see if our site-directed mutagensis worked. We will digest each BAMT sample (representing an individual colony) with SpeI.

BAMTmut

• 36.5μL H20
• 5μL Buffer 2 (vortexed quickly)
• 0.5 μL BSA
• 7μL ng/uL DNA
• 1μL SpeI

BAMTpreMut

• 38.5μL H20
• 5μL Buffer 2 (vortexed quickly)
• 0.5 μL BSA
• 5μL 58.8 ng/uL DNA
• 1μL SpeI

Ligation Protocol for connecting BSMT and SAMT to the terminator

BSMT

5 uL BSMT (66.5 ng)

5 uL B0015 (52.1 ng)

1 uL Ligase Buffer

.5 uL Ligase

SAMT

5 uL SAMT (50.5 ng)

5 uL B0015 (52.1 ng)

1 uL Ligase Buffer

.5 uL Ligase

Then, transformed four LB AMP plates with the two mixes (two plates of each) plus the positive control pUC19 in accordance with the normal protocol.

Repeat of smell test experiment for GC quantification

Cells with SAMT and BSMT were grown up with varying concentrations of Salicylic Acid in the media. The following tubes were made with 20 uL of the appropriate cells and 7 mL of the appropriate media: [note: all quantities in uM are really ug/mL]

SAMT in LB Kan with 17.36 uL SA (200 uM)

SAMT in LB Kan with 21.70 uL SA (250 uM)

SAMT in LB Kan with 26.04 uL SA (300 uM)

SAMT in LB Kan with 30.38 uL SA (350 uM)

SAMT in LB Kan with 34.72 uL SA (400 uM)

BSMT in LB Kan with 17.36 uL SA (200 uM)

BSMT in LB Kan with 21.70 uL SA (250 uM)

BSMT in LB Kan with 26.04 uL SA (300 uM)

BSMT in LB Kan with 30.38 uL SA (350 uM)

BSMT in LB Kan with 34.72 uL SA (400 uM)

Controls:

SAMT in LB Kan with 26.04 uL SA (300 uM) (Not Induced)

BSMT in LB Kan with 26.04 uL SA (300 uM) (Not Induced)

BL21 in LB with SA (not induced)

BL21 in LB (not induced)

MEETING: Project Descriptions

1. E. coli lab strain
   • We want to engineer a pleasant perfuming E. coli system that (with no exogenous action or input) can grow and smell when it reaches stationary phase
     • all substrates will be biosynthesized by cells
     • stationary phase controls gene expression
     • pleasant smell does not need to compete with indole's unpleasant smell
2. Methylobacteria and/or Pseudomonas fluorescens biofilter
   • combine our perfuming power into bacteria that already mop-up smelly compounds
   • colonize a biofilter with our engineered bacterial system

MEETING: Project 1: E. coli, Progress

1. Biobricks of SAMT, BAMT, BSMT, ATF1 sequence results have arrived
2. 1 smell is detectable in SAMT and BSMT cell cultures and GC quantification work is underway
3. pUC18-derivative plasmid pME3366 (for SA biosynthesis via genes pchBA) has arrived
   • liquid culture cells grew up successfully in LB Amp
4. 1 indole knock-out strain has arrived
   • liquid culture cells smell noticibly non-indole-ish
5. Stationary phase promoter primers have arrived
6. Isoamyl acetate ordered
7. Primers for the 1st gene in Isoamyl alcohol biosynthesis has been ordered

MEETING: Project 1: E. coli, To Do

1. Need to order primers for 2nd Isoamyl Acetate gene (waiting on Tom's advice)
2. Check that coding mutagenesis reactions were successful for BAMT
3. Repeat ATF1 transformation
4. Hook up each biobrick coding region to a Rbs, Promotor, and Terminator
   • Right now we are repeating ligation/transformation for the digest of coding region-terminator
5. Check if we successfully PCRed out the stationary phase promoter from the E. coli genome by using a shorter annealing time
6. Make competent our new indole-negative E. coli strain YYC912
7. GC quantify YYC912's lack of indole activity
8. Double transform pME3366 plasmid and Dudareva SAMT pET28-a(+) into Top10 cells (smell and make GC measurements of methyl salicylate)
   • try a similar transformation with our biobrick plasmids once complete
   • try a similar transformation with our indole-negative tnaA mutant E. coli cells (once made competent)
9. Continue email follow-ups, etc. and research!!

MEETING: Project 2: Methylobacteria/Pseudomonas, Progress

1. PWUBR is an E. coli to Methylobacteria shuttle vector
   • requested from J. Hubacek
2. Leisinger lab also has an interesting methyl-e.coli strain
   • requested from T. Leisinger
3. pUCP22 is an E. coli to Pseudomonas shuttle vector
   • requested from Herbert Schweizer
4. We have Pseudomonas fluorescens in our fridge

MEETING: Project 2: Methylobacteria/Pseudomonas, To do

1. Order an appropriate methylobacterium strain for lab work
2. learn everything about working with/growing methylobacteria and/or Pseudomonas
3. research biofilters and construction
   • search for more sources of similar vectors if we don't hear back soon on our requests