Team:UCLA/Notebook/Honeybee Silk/26 May 2015

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PCR of Honeybee G-Block

• Diagnostic PCR, will be running a temperature gradient
  • From the NEB Calculator, I found the TM Melting temperature to be 67C. I will also run two samples at 64 C and 70C, then compare on a gel.
• Cloning AmelF3 to prepare for insertion into pET24a expression vector
  • Primers 10 and 11
  • See Benchling for reference

Component	Volume (out of 25uL)
5X Q5 Reaction Buffer	5uL
10mM dNTPS	0.5uL
10mM primer 10	1.25uL
10mM primer 11	1.25uL
Template (Honeybee G-block)	1uL
Q5 High Fidelity DNA Polymerase	0.25uL
Nuclease Free Water	16.25uL

Program

Step	Temperature	Time
Initial Denaturation	98C	3 min
Cycles (x25)	98C	10s
Annealing	64/67/70C	20s
Extension	72C	30s
Final Extension	72C	2min
Hold	12C	Hold

Gel Visualization

I ran all three samples on a 1% agarose gel against a 1kB ladder. Expected BP length: ~1000bp

Results: All three bands were present at the correct bp length, the cleanest band was from the 67C annealing temperature.

Gel Image

BugBuster Protocol

• Using the Bugbuster 10X cell lysis reagent in order to purify our honeybee silk protein via inclusion bodies.
  • Based protocol off of the Novagen manufacturer's protocol which can be found [http://wolfson.huji.ac.il/purification/PDF/Protein_Expression_Extraction/NOVAGEN_BugBuster_protein_extraction.pdf here].
• Expressing honey bee silk in E. Coli, from [http://parts.igem.org/Part:BBa_K1763001 BBa_K1763001]
  • See 5/18 and 5/19 for details on the protein expression.

Step by Step Protocol

1. Determine wet wet weight of cell pellet after spinning liquid culter at 10000 x g for 10 min.
   1. 2.5 g
2. Resuspend in 5 ml/g Bug Buster (1x) by pipetting and gently vortexing.
   1. This is 12.5 ml in our case
3. Put on shaker or rotating mixer for 15 min at RT
   1. Took our first fraction at this point of the full cell lysate (F1)
4. Centrifuge 16000 g 20 min at 4 degrees C
   1. Took next fraction at this point of the supernatant, labeled (S1)
5. Resuspend pellet in same volume of 1X bugbuster as before
   1. 12.5 ml
6. Pipett up and down and vortex gently to get an even suspension.
   1. Took third fraction at this point (F2)
   2. I did not go to great lengths to get an even suspension here, but I noticed in retrospect that the protocol emphasized the importance of this in order to get pure inclusion bodies.
7. Add dry lysozyme to final concentration of 200 ug / ml
   1. For 12.5 ml solution I added around 2.5 mg of lysozyme
   2. I did not add any DNAse because it was not mentioned on the manufacturer's protocol, but in the future I will try adding 1 ul of DNAse at this point because chromosomal DNA might have been a problem in subsequent steps.
8. Add 6 volumes of 1:10 diluted bugbuster (.1X)
   1. At this point we split the solution up into two 50 ml falcon tubes and added 37.5 ml of .1X bugbuster to each tube
9. Centrifuge 16000g 15 min. 4 degrees C to collect inclusion bodies.
10. Remove supernatant w/ pipett, take next fraction (S3)
    1. After spinning this down, there was a pellet, with an opaque and viscous substance. (Im guessing it might be chromosomal DNA)
    2. It was difficult to remove the supernatant because the viscous liquid kept getting sucked up and bringing the pellet up with it.
11. Resuspend pellet in 1/2 volume of original 0.1X bug buster solution
    1. 18.75 ml per tube
12. Mix to get an even suspension by pipetting and vortexing for several minutes and spin down as in step 9.
13. Repeat step 11-12 two more times.
    1. Take samples of supernatant at each wash step
14. Resuspend inclusion body pellet in 3% SDS solution and incubate at 60 C in the water bath for 2 hours
    1. I used around 2 ml, but I probably could have used less, because the pellet dissolves well in the heat
15. Store solution at 4C until further required.
    1. I noticed that after a day in the 4C, the solution gelled up.
    2. According to protocol, storage at temperatures below 4°C may cause precipitation of the detergents in BugBuster reagent. Incubate in a room temperature water bath with gentle swirling to redissolve.

Some Notes

• I collected the viscous (potential chromosomal DNA) substance and labeled it S4.
• After the second wash step, I tried to remove all of the viscous substance, after which the resulting pellets were much smaller, and difficult to break up even after several minutes of vortexing.
• Next Step is to run the different fractions, and the dissolved purified silk on an SDS gel to see if I got any protein.

Gel Image

Fig. 1 Expected size of product is 31.6 kDA

• The product was expected to be purified in the lane labeled inclusion body.
• The bright band in the inclusion body lane seems to be around 16 kDA as opposed to the expected 31.6 kD.
• Im not sure what to make of this data. Although there is a bright and somewhat isolated band, it is not the right size.

Might have something to do with the fact the that the sample was a few weeks old when it was run.

• I will try this experiment again using a T7 promoter system.