Team:UCLA/Notebook/Recombinant Expression/12 August 2015

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TO-DO LIST

1. Inoculate 1L of LB supplemented with 1mL of 1000x chloramphenicol with MaSP2 15-mer starter culture (once the culture hits an OD600 of 0.6-0.8).

2. Once the 1L hits an OD600 of 0.6-0.8, induce with IPTG solution at a final concentration of 0.5 mM (5mL of a 100mM stock).

3. Take 1mL expression time point samples before adding IPTG, and 1 hour after adding IPTG (Before IPTG, 1hr, 2hr, 3hr, 4hr). Centrifuge and harvest the cells at the 6 hr mark.

4. Centrifuge the cells at 5,300g for 15 minutes at 4 degC. Remove medium, rinse cells with 8mL of MilliQ water, and re-centrifuge at 3,300g for 15min at 4degC. Remove water, and weigh pellet (make calculations on lysis steps here).

5. Resuspend the cell pellet at 1:3 (wt/vol) ratio and store at -20 degC. Prepare reagents for lysis tomorrow.

6. Run SDS PAGE with samples of white mystery, pelleted fraction (resolubilize in SDS), cleared lysate, and the 3 samples from the 12-mer expression for time point analysis. (Use the GTE-LSB-Boil method).

7. Warm the clear cell lysates to 37degC, and add 3 uL of DNAseI to each of the lysates, rocking them on a shaking platform at RT for 30 minutes. Observe to see if viscosity is degraded. If not, incubate for longer or add more DNase I.

8. After degradation, set up a column as described by Teule et. al. 2012 to conduct the IMAC purification. Save fractions for SDS-PAGE on Thursday.

9. Conduct a BCA assay on the IMAC fractions using MicroBCA protocol to analyze samples immediately (at least, before ether meeting with Sri).

9. ORDER: (1) SDS-PAGE Gels (Bio-Rad 4-20% TGX Precast Gels), (2) Deoxycholic Acid (VWR, Sodium Salt), (3) IPTG from GoldBio. Give to DiCarlo for signature ASAP.

Starture Cultures

Machine needs to be recalibrated.

== SDS PAGE of samples ==:

Supernatants were heat treated at 60 deg Celsius for 10 minutes before adding 3 uL of DNAseI to each solution, and shaking RT 350rpm on a shaking platform. Insoluble pellet fractions were treated with 10% SDS to solubilize the insoluble proteins. White Pellets were solubilized using 10% SDS as well. Cells were resuspended in 50uL of 1X LSB and boiled for 10 minutes at 95deg, and spun down RT 10min at max speed.

SDS PAGE --> Ladder - 9mer White Pellet, 12 mer White Pellet, 9mer pellet, 12-mer pellet, 9-mer cleared lysate, 12-mer cleared lysate, 12-mer 2hr, 12-mer 3hr, 12-mer 4hr

20 uL of each buffered sample were loaded.