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

Hey guys...I came in at 7.15 and put the plate, liquid cultures and colony PCRs in the 4C fridge (look at the bottom of the fridge for all of this stuff). I went to sleep at the student center and should be in soon. -André

rpos Promoter Update

I plotted fluorescence/OD600 vs. OD600 and saw that the rate of expression of the promoter and the control went down with increasing cell density. I also plotted fluorescence vs. OD600 and saw a rough increase with cell density for both the promoter and control. These are unexpected (and sad) results, but for now, we will move on to the osmY promoter as that seems to have a more pronounced difference in expression in the stationary phase.

Ran gel on the colony pcr products

Ran a gel with the following lanes (left to right):

• 2-log ladder
• SAMT colony A pcr product (expected length 1471bp)
• SAMT colony B pcr product (expected length 1471bp)
• SAMT colony C pcr product (expected length 1471bp)
• SAMT colony D pcr product (expected length 1471bp)
• BAMT colony pcr product (expected length 1414bp)
• ATF1 colony pcr product (expected length 1897bp)

Made glycerols of cells with biobricks

Made 6 glycerols from the liquid cultures: SAMT-A, SAMT-B, SAMT-C, SAMT-D, ATF1, and BAMT. The cells in these should have the biobricked versions of our genes (backbone: pSB1A3-1)

Miniprep

Did minipreps to isolate the biobricked plasmids. Nanodrop concentration checks yielded the following concentrations:

• SAMT-A: 60.4 ng/μL
• SAMT-B: 84.5 ng/μL
• SAMT-C: 66 ng/μL
• SAMT-D: 59.7 ng/μL
• ATF1: 67.2 ng/μL
• BAMT: 68.2 ng/μL

osmY Promoter Primer Design

Truncated Forward Primer: 5'- GTT TCT TCG AAT TCG CGG CCG CTT CTA GGCT TAT GTT TTC GCT GAT ATC - 3'

Total Length: 49 bp

Annealing Length: 21 bp

GC Content: 38.1%

Melting Temperature: 49.5 degrees C

hairpin deltaG: -2.66 kcal/mol

self dimer deltaG: -99.97 kcal/mol

Full Forward Primer: 5'- GTT TCT TCG AAT TCG CGG CCG CTT CTA GCTG GCA CAG GAA CGT TAT C - 3'

Total Length: 47 bp

Annealing Length: 19 bp

GC Content: 52.6%

Melting Temperature: 53.4 degrees C

hairpin deltaG: -3.99 kcal/mol

self dimer deltaG: -98.3 kcal/mol

Reverse Primer: 5'-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TGT TAA ATA TAG ATC ACA ATT TTG- 3'

Total Length: 54 bp

Annealing Length: 25 bp

GC Content: 20.0%

Melting Temperature: 46.4 degrees C

hairpin deltaG: -2.86 kcal/mol

self dimer deltaG: -99.44 kcal/mol

heterodimer deltaG with Forward Primer: -99.44 kcal/mol

heterodimer deltaG with Conserved Forward Primer: -99.97 kcal/mol

To do in afternoon

1. design sequencing primers for pSBIA3-1 biobrick backbone - or just use VF2 and VR:)
2. send BAMT_BB, BSMT_BB, SAMT_BB, and ATF1_BB off for sequencing analysis
3. run JMT PCR with all cDNA libraries that we have in lab currently
4. smell methyl jasmonate in hood
5. email non-responders to follow up on requests (Kate)
6. GC at 4 pm (Stephen and Veena)

JMT PCR

1. CD4-34
   • 49 μL of Tom's PCR mix
   • .6μL of each JMT primer (forward and reverse)
   • 1 μL of CD4-34 cDNA library
2. CD4-14, CD4-15, CD4-34
   • 49 μL of Tom's PCR mix
   • .6μL of each JMT primer (forward and reverse)
   • 1 μL of CD4-14, CD4-15, and CD4-34 cDNA libraries
3. CD4-34 ZapII
   • 49 μL of Tom's PCR mix
   • .6μL of each JMT primer (forward and reverse)
   • 1 μL of CD4-34 ZapII cDNA library
4. BSMT (+ control)
   • 49 μL of Tom's PCR mix
   • .6μL of each BSMT primer (forward and reverse)
   • 1 μL of BSMT template
5. Run the tube on the thermocycler at: 95deg for 3:00, then cycle through: (a) 94deg for :30 (b) 55deg for :30 (c)68deg for 2:15, then 72deg for 10:00

SEQUENCING

1. send in for analysis: BAMT_BB, BSMT_BB, SAMT_BB, and ATF1_BB
2. use: VF2 and VR
3. notes:
   • Add 180ng of template DNA
   • Add 3.2 pMoles of primer (0.128 uL from 25 uM working solution)