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Transformation with the Silk+GFP Construct

1. Thaw cells (~80uL) on ice for 10 minutes.
2. Add 1uL of ligated product.
3. Place on ice for 5 minutes.
4. Rescue in 320uL of SOC, incubate and shake for 1 hour.
5. Warm chloramphenicol plates at 37C.
6. Spin down bacteria to pellet, discard supernatant.
7. Make 2 serial dilutions (1:10 and 1:100)
8. Plate 100uL of bacteria (1:1, 1:10, 1:100) on each plate.
9. Spread with beads, incubate at 37C.

Multiple Mini Expressions: Optimizing protein yield

• Today we are testing multiple growth conditions of our bl21 cells for total cell and protein yield. Parameters we are optimizing are iptg concentration, and growth temperature.

• We will test these different conditions using the following protocol

1. Grow up a starter culture using glycerol stock #1 of our silk in BL21 to an OD of around 1. (4 to 5 hours)
2. Add 50 ul of starter culture to 5 ml of LB starting around 5 pm along with 5 ul of Kan.
3. Add in the varying amounts of IPTG and use the two different temperatures.

• Use the following protocol to analyze the cell pellet.

1. Record the weight of the pellets.
   • In total, the pellet weighed 1.75 grams, or about 0.6 grams per 100ml, which is lower than the 0.8 grams we have gotten in the past.
2. Transfer pellet to one falcon tube.
3. Resuspend in 5 ml/g (8.75ml) of pellet Bug Buster (1x) by pipetting and gently vortexing.
4. Put on shaker or rotating mixer for 15 min at RT.
   • Take first fraction, (F1) of this full cell lysate.
5. Centrifuge 16000 g 20 min at 4 degrees C
   • Take second fraction of this supernatant (S1) which should contain soluble proteins, while our desired protein should be in the pellet.
6. Resuspend in the same volume of 1X Bugbuster 8.75ml as above.
   • Make sure the pellet is completely resuspended!
   • Take Fraction at this point (F2) Cell lysate #2
7. Add DNAse (1 ul) and let rotate 20 min. (This may have been too much DNAse but it should be ok.)
8. Add dry lysozyme to concentration of 200 ug / ml
   • Dissolved the lysozyme in water and added the water.
   • Let incubate on ice 30 minutes, swirl every 5 min.
9. Take another fraction (F3) here, to see if there is a difference after adding the DNAse and the lysozyme.
10. Add 6 volumes of 1:10 diluted bugbuster (.1X)
    • Can split up into two falcon tubes if necessary.
    • Vortex for 1 minute
11. Centrifuge 16000g 15 min. 4 degrees C to collect inclusion bodies.
12. Collect supernatant as fraction (S2). Inclusion body should be the pellet.
13. Resuspend inclusion body pellet in 3% SDS solution and incubate at 60 C in the water bath for 2 hours.
    • Used 10 ml of SDS. (Final)
14. Store at 4C for further processing and analysis.

Results

• The results of this experiment was intended to guide which growth conditions we use in order to express honeybee silk and conduct further experiments.
• The results we are using to determine the optimal conditions are the weights of the cell pellets, the results of the BCA assay and the SDS PAGE.

• Keep in mind that these weights are not super accurate because they are the wet weights, and we just tried to get as much water out as possible.
• SDS PAGE gel

• The BCA assay also did not indicate much of a difference between different IPTG concentrations.
• Therefore I think I will just continue with 37 C and 0.5mM iptg for the time being.