ResearchPage+-+Joshua

Joshua's Research

12.09.11

Reaction condition E absent from graph above because it was spilled.



Week 14:





Week 13:



Week 12:











Week 11:
 * Of the 30,000 ligands in the MW set, I have tested ligands 2,001-18,000. None of these ligands yielded higher fitness scores than the ligands we chose to order. The top two ligand scores obtained from the first 2,000 ligands (sceened last week) were 98.66 and 88.14. The top five scores obtained this week (ligands 2,001-18,000) are listed below.



Week 10:



Week 9:
 * Grew up 1.5L of LB + BL21 and most likely ruined the protein
 * Grew up 3L of LB + BL21 inducing after 0.8 OD600. This value was reached 1hr 40min after addition of overnight growth. Next time, will use less overnight growth in 1L LB samples in order to catch an OD600 of 0.3.

Week 8:
 * FPLC purified MptpB
 * Concentrated and obtained the following readings






 * First inhibition assay



Week 7:
 * Out

Week 6:
 * Lysed two bacterial pellets
 * Purified 2 samples independently (approx. 15 mL each)
 * Ran a gel using the sample that had a concentration 0.19 mg/mL




 * Concentrated Elution #1 and Wash for FPLC purification. A significant amount of MptpB was lost in the wash step.

- Joshua - avoid putting links to files here. Show results in a table or in a screenshot instead. -- Dr. B

Week 5:
 * Worked on Virtual Screening

Week 4:
 * Worked on PyMol

Week 3:
 * Transformed BL21 cells using the good, midi prepped plasmid sample
 * Picked two colonies, one from each of two plates, and grew up two samples for 12 hr overnight
 * Began to grow up protein (two 550mL samples of BL21 cells + LB + Kan left on the counter for 1 hr)
 * Picked a colony from the plate that had 10 uL of transformed plasmid plated (colonies are bigger and more spread out as opposed to the plate that had 50 uL of plasmid plated) - trial 2 of the protein expression scale up
 * Pelleted two samples of BL21 bacteria (trial 1 samples had a wait period at room temp of 1 hr before IPTG was added and the trial 2 sample didn't have an exponential growth pattern)
 * Scaled up bacteria and grew up MptpB once again (trial three) using 1M K3PO4, 10% glycerol with LB + Kan for the overnight growth of one sample. For the second sample I re-suspended the bacteria in fresh LB + Kan after growing it up in LB + Kan (centrifuged BL21 cells at 6000g for 5 min). The 10% glycerol was used as an extra energy source and the K3PO4 was used as a buffer since the 50mL of LB from overnight growth becomes acidic. For one sample I simply re-suspended the bacteria in fresh LB.



Week 2:
 * Transformed DH5 alpha cells using the plasmid sample containing my GOI after it's been mini prepped




 * Grew up transformed DH5 alpha cells and midi prepped them
 * Sent two samples to sequencing one of which had a 100% match with my GOI

Week 1:
 * Confirmed a 100% match for my GOI in pNIC-Bsa4


 * Good pNIC-Bsa4 + MptpB gene read after Midi Prep**:



Transformation Efficiency:



























The top band matches up with the secondary PCR product. However, there appears to be another smaller band. The forward and reverse primers were probably used to amplify both strands. I continued with the cloning procedure despite the fact that my PCR^2 product appears to contain two amplified sequences.



The lower band appears much brighter than the upper (desired) band after PCR cleanup. Some of the MPTPb coding sequence may have not been eluted properly.

The pNIC-Bsa4 fragments don't appear because I mistakenly used 2.25 ng when treating with BsaI rather than 2.25 ug, the amount called for in the protocol for pNIC-Bsa4 cloning.