Team:Caltech/Week 2

Monday 6/21

• Transformed additional bricks from distribution with other antibiotic resistances onto plates & liquid culture.
  • I15008, I15009, I15010, E0033, M30109, K112000, J09250

Tuesday 6/22

• Transformation success is ambiguous - the plates are covered in a lawn of bacteria, apparently indicating an issue with the antibiotic selection. This implies either a problem with the stock solution of antibiotic (Kan/Strep), or the procedure of plating the antibiotics on the surface of the LB-agar plates.
  • To find out what happened, we set up a simple experiment for both ampicillin (amp) and streptomyocin (strep):

Take 3 LB-agar plates with no antibiotic and plate 50uL of 1000x, 10x, or 2x antibiotic stock solution on each, spreading with sterile glass beads. Let the plates dry for 1 hour at RT. Additionally, prepare an LB-agar plate containing the desired antibiotic in the agar. Split all four plates in half and streak each plate with both a cell strain that should be resistant to your antibiotic, as well as a strain the should not be. Incubate the plates overnight at 37C.

If the antibiotic was prepared correctly, and is present in high enough concentration on the plate, only half of the plate should have any colonies present: the side with the desired resistance. Plates with cells on both sides do not have enough antibiotic; those with no cells have too much.

• Re-performed the transformations on two of the bricks & plated on LB-agar plates with the antibiotic incorporated.

Wednesday 6/23

• Transformation plates are still covered in a film of bacteria. The negative control for the kanamycin test grew bacteria, suggesting that there is an issue with the kanamycin stock, which we will have to remake.
• The amp & strep antibiotic test plates indicate that plating 50uL of 1000x stock is sufficient for proper bacterial selection:

Thursday 6/24

• Our NEB Biobrick assembly kit arrived today! We began digesting & ligating bricks to assemble our light induction/lysis construct.
  • Desired construct: R0082 (light-induced promoter) + B0034 (RBS) + K124017 (lysis gene cassette) + B0015 (terminator) in a non-amp/non-cm backbone (since our light transduction brick, M30109, is A/C resistant).
  • Began five digests according to the NEB Biobrick assembly kit protocol:
    • R0082 (us)
    • B0034 (ds)
    • K124017 (us)
    • B0015 (ds)
    • Cm linear backbone (pSB1C3)
  • Began two ligation reactions according to the Biobrick assembly kit protocol:
    • R0082/B0034/pSB1C3
    • K124017/B0015/pSB1C3
  • Transformed the ligation products into DH5alpha (Invitrogen) competent cells via heat shock and plated (incorrectly) on LB-amp plates.

Friday 6/25

• Re-transformed the ligation products from 6/24 into DH5alpha cells via heat shock and both: plated 100uL of cells on LB-cm plates and added remainder to create LB-cm liquid culture. Both were incubated overnight at 37C.

Weekend 6/26-27