Team:Nevada/Week 13

August 13

Joe:

Primer stock- RFP f-anti

PCR of [f]SBP-promoter to RFP anti

Michelle:

Transformation of Friday’s ligation of SBP (SpeI and PstI)-LRP (PstI and XbaI) digested with

SpeI and PstI with Terminator (PstI and XbaI) and Promoter (TETrbs) (EcoRI and SpeI) into

Top 10 competent cells onto KAN plates.

Miniprep colony M2 from transformation of PCR Fusion SBP (SpeI and PstI)-LRP (PstI and XbaI) followed by PCR ::::purification with glass milk because of mistake in miniprep protocol.

Nanodrop miniprep of colony M2 PCR Fusion SBP (SpeI and PstI)-LRP (PstI and XbaI) before transforming into BL21 ::::competent cells and onto AMP plates.

Justin and Dafne

Culture successful colonies from last weeks ligation in LB-amp until OD reaches 0.6

Add IPTG in varying concentrations

Tube 1: 0.01 uM Tube 2: 0.1 uM Tube 3: 1.0 uM Tube 4: 10.0 uM

After 4 hrs, pellet, place in -80 freezer

After 8 hrs, pellet, place in -80 freezer

Transform SBP-B12 with L-arabinose promoter into BL21 cells

Jeremiah & Chris:

Miniprep colony 2

Transform purified plasmid into BL21 expression cells

Attempt to express the protein while in TOP10 cells using an IPTG gradient

Took a 3 hour and overnight time point

Jeremiah & Chris:

Miniprep colony 2

Transform purified plasmid into BL21 expression cells

Attempt to express the protein while in TOP10 cells using an IPTG gradient

Took a 3 hour and overnight time point

August 14

Michelle:

Check yesterday’s transformation of SBP (SpeI and PstI)-LRP (PstI and XbaI) digested

with SpeI and PstI with Terminator (PstI and XbaI) and Promoter (TETrbs) (EcoRI and

SpeI).

Transformation yesterday of colony M2 from transformation of PCR Fusion SBP (SpeI and PstI)-LRP (PstI and XbaI) on :::AMP plates was wrong. Needed to be transformed onto Ampicillin-Chlorophenicol (AMP-CM) plates.

Re-do transformation into BL21 competent cells onto AMP-CM plates.

Justin and Dafne:

Culture SBP-B12 in BL21 cells to an OD of 0.6

Add concentrations of L-arabinose as done previously, also adding 5 nM Vitamin B-complex

Take time samples as done above

Jeremiah & Chris:

Run the time points on a polyacrylamide gel

August 15

Michelle:

Check transformation of colony M2 from transformation PCR Fusion SBP (SpeI and

PstI)-LRP (PstI and XbaI) with BL21 competent cells on AMP-CM plates.

Culture smallest colony from this transformation, which we will call M2-BL21 with LB AMP-CM broth.

Obtain pellet and supernatant preparation from 8ml of colony M2 from transformation of PCR

Fusion SBP (SpeI and PstI)-LRP (PstI and XbaI) not IPTG induced for Western blot transfer.

Justin and Dafne:

Perform western blot analysis of both protein expression experiments

IPTG unsuccessful, L-arabinose induced SBP-B12 in BL21 cells very successfull

Jeremiah & Chris:

Western blot protocol

Cultured BL21 colony from transformation plate

August 16

Michelle:

Western blot transfer continued for pellet and supernatant samples of colony M2 from

transformation of PCR Fusion SBP (SpeI and PstI)-LRP (PstI and XbaI) not induced with

IPTG and membrane developed.

August 17

Jeremiah & Chris:

Developed western blot of expression in TOP10 cells

No expression was detected (took pictures of the membranes on my iphone)

Attempt to express the protein while in BL21 cells using an IPTG gradient

Took a 3 hour and overnight time point

Cultured same BL21 colony with thiamine supplement added

Michelle:

Western blot transfer of 8ml from culture to obtain pellet and supernatant samples of smallest

colony from transformation of M2-BL21 from 8/15.

Jeremiah & Chris:

Run time points on a polyacrylamide gel (BL21 cells with no thiamine added)

Attempt to express the protein while in BL21 cells using an IPTG gradient (BL21 cells with thiamine added)

Took a 3 hour and overnight time point