IGEM:MIT/2006/Notebook/2007-1-18

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To Do:

1. Submit all 108 sequencing samples- DONE

2. Check sequencing results from 1/17 and 1/18- DONE

3. Check antibiotic tests of osmY.E0840 SN, osmY.inverter.E0840 FN, R0040.E0840, B0015 (control), and the indole-knockout cells- DONE

4. Retransform osmY.E0840 FN prep

5. Digest osmY.inverter.J45119 and J45320- DONE

6. Ligate osmY.inverter.J45119 to J45320- DONE

8. Obtain Top 10 Competent Cells- DONE

9. Ligate inverter.E0840 and osmY SN structures- DONE

10. Transform osmY.inverter.J45119, osmYSN.inverter.E0840, and mixes of premade J45800 hopefuls

11. Find protocol used for "good" plate reader experiments- DONE (see http://openwetware.org/wiki/IGEM:MIT/2006/Notebook/2006-8-12)

12. Touch base with GC people

Results from Antibiotic Tests

R0040.E0840

SUCCESSFUL (grew on AMP and not on AT, AK, or AC)

osmYFN.inverter.E0840

SUCCESSFUL (grew on AK and not on AT or AC)

osmYSN.E0840

SUCCESSFUL (grew on AT and not on AK or AC)

B0015 (Control)

SUCCESSFUL (grew on AK and not on AT or AC)

Indole-Knockout cells

SUCCESSFUL (grew on C but not on A, T, or K, consistent with Yale's listing of the strain as Chloramphenicol resistant)

Sequencing Results

The osmY.inverter.J45119 structure from the fridge was correct along with all of the J45120s which passed the antibiotic test. Thus, we will continue to use "J45120 A" as we used in the construction of a new J45700.

All of the premade pBANANA structures had either bad results or failed sequencing. The only thing we can say for sure is that one of the structures has J45400 in it. I believe that our best bet is to continue with the J45600 and pBANANA structures I have constructed this past week since we were able to smell bananas in both structures.

  • NOTE- The osmY.inverter.J45119 structure has the short version of the osmY promoter in it. J45250 has the long version of osmY in it. This was part of our original, optimistic plan to have less expression of mint, so that the banana smell could overpower residual methyl salicylate in stationary phase. To make these versions match our fluorescent structures, I will retransform an osmY.E0840 full version today and digest osmY short and inverter.E0840 today to ligate and transform tomorrow. Right now, we have osmY.E0840 short and osmY.inverter.E0840 long.
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