January2016/Experimental design

We're basing these experiments on the Kim2015 paper.

These are the experiments we expect to run:

• Ligate synthesized DNA into linearized plasmid
• Transform plasmids into ecoli (DH5 alpha)
• Stick extra DNA in TE buffer and freeze it
• Grow up liquid culture and make glycerol stock
• Do plasmid prep and put in freezer on TE buffer
• Sequence plasmid
• (optional: try to purify protein from e. coli and run gel)
• Transform plasmid into yeast
• Grow yeast on selective media and make glycerol stock
• Grow yeast in bioreactor and induce expression of our protein
• Run nickel column purification of extracellular media
• If the above doesn't work, try to lyse cells and repeat purification
• Run page gels of purifiction product

PLEASE HELP BY WRITING UP OR LINKING THE NECESSARY PROTOCOLS FOR THE ABOVE EXPERIMENTS

For RVC protocols in use until 2015, see General Protocols section of Shared Laboratory Notebook

Create and prepare plasmid constructs

We need to ligate our synthesized DNA into the DNA 2.0 plasmid.

Supplies and reagents

• DNA 2.0 plasmids pD1204
• Our synthesized DNA from IDT: Both hCMP and full bovine
• Electra Cloning Reagents Kit (already ordered from DNA 2.0)
• ddH20 (at least 30 microliters)

Protocol

We're using the Electra System from DNA 2.0 which uses SapI sites. The protocol is here.

Note: Electra Buffer should be aliquotted to avoid multiple freeze thaws!

Experimental design

1. Dissolve dry pD1204 plasmid DNA pellet in TE buffer
2. Dissolve dry pJ1204-03C control plasmid DNA pellet in TE buffer

For each of the two IDT sequences:

1. Dissolve dry DNA pellet from IDT in TE buffer
2. Complete Electra Cloning Protocol using IDT sequence + pD1204 plasmid

Long term storage of unused DNA

Any remaining DNA from the previous step should be stored in case we want to re-do the experiment or try something else later.

Supplies and reagents

• TE buffer (10 mM Tris-Cl, pH 7.5; 1 mM EDTA)
• Our excess DNA from previous step
• At least 9 cryotubes

Experimental design

Storing in TE buffer at -20C performs best - see Evaluation of DNA Plasmid Storage Conditions

Freeze/thaw cycles can damage the DNA, so best would be to store small single-use aliquots.

Transform plasmids into e. coli

Supplies and reagents

This is what the Open Insulin group did, as advised by an expert.

• Our plasmid constructs: pD1204-hCMP, pD1204-BK (bovine kappa-casein) and control plasmid pD1204-03C
• Competent E. coli (BioCurious has an excess that we can use)
• Antibiotic that the plasmid has resistance to
• For making 20 LB agar plates
  • 5mL Transformation buffer (10% PEG 8000, 5% DMSO, 25mM MgSO4, 25mM CaCl2, LB)
  • 18g LB Amp Agar or LB Kan Agar
  • 20 plates
  • 500mL glass jar
  • 10 inoculation loops
  • 50 - 1.5mL microtubes

Protocol

• Open Insulin group's protocol

• • 18g of LB Agar should pour ~20 plates from 500mL
  • Add 100uL transformation buffer to a microtube
  • Using an inoculation loop scrape enough bacteria to almost fill the loop and mix it into the transformation buffer. Do this twice
  • Add 100ng-500ng of plasmid to the microtube containing the transformation buffer and place at 4C or in the fridge for 30 minutes.
  • “Heat Shock” the bacteria by placing them in water that is ~42C for 30 seconds and then let them sit at 37C(room temperature is fine if you don’t have a 37C incubator) for 1-3 hours (in this step the bacteria that have accepted the DNA will produce antibiotic resistant proteins for selection and replicate hopefully creating more bacteria with the plasmid)
  • Take 100uL of your transformation and put it on a LB agar plate with the correction antibiotic to select for your plasmid
  • Incubate plate overnight at 37C or ~24 hour at room temperature(RT)(if using the pVIB.pJE202 bioluminescent plasmid incubate only at RT)

Other protocols we can use:

• Bacterial Transformation of DH5a
• Transformation of plasmid DNA to competent E. Coli cells
• Dh5-Alpha Competent E. Coli User Manual
• Plasmid Transformation into DH5alpha E.coli cells using Heat Shock
• Protocol: Transformation of Plasmids/Cosmids into E. coli
• http://openwetware.org/wiki/Transforming_chemically_competent_cells]

Experimental design

Complete transformation protocol in duplicate or triplicate for each construct. In addition complete the protocol with no plasmid (negative control and with neither e. coli nor plasmid (contaminant check).

Grow up liquid culture

Once we have successful transformants we will need to pick colonies and grow them up in LB+amp.

Supplies and reagents

• Our transformants
• 37 C incubator with shaker
• LB+amp broth

  To make LB+Amp broth, mix 25g/L LB-Miller powder in distilled water, autoclave for 20min at 15psi, cool to 55C, add 50ug/L Ampicillin (or 1ml/L of 1000x Amp stock solution)

• Tubes (size not important)

Experimental design

Pick successful transformants from each non-control LB+amp plate (assuming controls turned out as expected), dissolve in 37 C LB+amp tube and grow overnight in shaker in 37 C incubator.

Make glycerol stock

For long term -80 C storage we need to make glycerol stocks.

Supplies and reagents

• LB+amp culture grown overnight with our transformants (from previous step)
• Glycerol
• dH20
• Cryotubes

Protocols

• Glycerol Stock protocol

Experimental design

For each of our transformants make at least three cryotubes containing a 50/50 mixture of autoclaved glycerol and the LB+amp with the transformants. Label it and shove it in our -40 C freezer (or -80 C if we have it operational).

Extract plasmid from liquid cultures

Supplies and reagents

• LB+amp culture grown overnight with our transformants (from previous step)
• BioMiga Plasmid MiniPrep kit 1
• 10,000 RPM centrifuge

Protocols

• BioMiga Plasmid MiniPrep Kit 1 protocol

Experimental design

We need to extract enough plasmid from each of our samples for the following three purposes:

• Sequencing
• Long term -20 C storage on TE buffer
• Short term 4 C storage for upcoming experiments

Prep and send DNA in for sequencing

ToDo: Find a sequencing company that is cheap (and preferably local) and see how we need to prep our sample before sending it in.

Alan recommends Genewiz - $7 per sequence, with overnight processing! I thought he mentioned they have a drop box locally, but I'm not sure where. They also have a list of Free universal sequencing primers, and there may very well be one on there we can use.

It looks like we can just submit the plasmid, and tell them which sequencing primer to use. Or do we need to do our own PCR on the plasmid first, and then submit the PCR product?

Transform plasmid into yeast

Last year we use Clontech's Quick & Easy Yeast Transformation

Quick & Easy Yeast Transformation Mix Protocol-At-A-Glance (for S. cerevisiae)

Here's some other yeast transformation kits we could use.

Yeast electroporation protocols

• What's electroporation? https://en.wikipedia.org/wiki/Electroporation

• http://www.umich.edu/~mapplab/protocols/yeast_transformation.pdf

  Sorbitol and LiTE

• http://openwetware.org/wiki/Wittrup:_Yeast_Transformation

  Tris/DTT; E buffer (Tris/sucrose/MgCl2)

• http://www.ncbi.nlm.nih.gov/pubmed/9605506

  LiAc and DTT; claims 2-3 orders of magnitude improvement

• http://peds.oxfordjournals.org/content/early/2010/02/03/protein.gzq002.full

  LiAc and DTT, plus sorbitol and CaCl in electroporation buffer

• http://www.geguchadze.com/PDF/protocols/CPonline/Doc/17901-17901.html

  LiAc and DTT, then sorbitol

• http://www.ncbi.nlm.nih.gov/pubmed/12684838 http://www.tandfonline.com/action/showPopup?citid=citart1&id=T4&doi=10.4161/bbug.1.6.13257 http://www.ncbi.nlm.nih.gov/pubmed/10870100

  Freezing in sorbitol and Ca at -80C improves electroporation after thawing

• http://www.tandfonline.com/doi/full/10.4161/bbug.1.6.13257#_i2

  Nice overview of the above methods

Grow yeast in bioreactor and induce expression of our protein

The plan is to grow our strain in a bioreactor. We'll need high aeration throughout the experiment.

The starting conditions for flask cultures used in the paper are:

• 2% yeast extract (we'll need defined media with no uracil)
• 2% glucose
• 3% galactose

This is likely fine for our initial proof of concept. Figure 2b from the Kim2005 the paper is especially helpful in understanding the flask cultures growth.

Bioreactor measurements

Biomass

The cell concentration was measured by a spectrophotometer at 660 nm (Lambda 20, Perkin-Elmer, USA). -- Kim2015 paper

We have a working spectrophotometer that can handle 660 nm at both CCL and Biocurious :)

Glucose / Galactose concentration

Glucose, galactose, glycerol, and alcohols in the culture supernatant were analyzed at 50°C using a high-per-formance liquid chromatograph (1100 series, Agilent Technologies, USA) equipped with a Shodex-SH1011 packed column (/ 8 mm·300 mm, Showa Denko K.K., Japan) and a refractive index detector. -- Kim2015 paper

This equipment would cost around $15,000 so not within our price range.

We could get a GlucCell Glucose Monitoring System which is the same type of device used by diabetics but created especially for cell cultures. I wonder if there is a real difference or if it's just a marketing gimmick. If we buy one we should test it against a normal drug-store blood glucose meter. It can be bought for $360 and the test strips are just over $1 each and sold in packs of 50.

On its website the GlucCell is compared to the NOVA BioProfile Biochemical Analyzer so that's likely an industry standard analyzer. An older 200 series NOVA can be found on ebay for $400 in allegedly working condition and with a 6 month warranty, but it is unclear if it will need reagents/chemistry to function. I expect that it will since the product page for the newer 400 series lists the glucose test methodology as enzyme/amperometric. It is possible that it is using probes with fixated enzymes but I wonder what the lifetime on such probes would be. Also the manual is not available anywhere that I can find. Someone could contact the company and ask if they still sell reagents fro the 200 and which reagents/supplies we'd need. If it _doesn't_ need reagents then that would be a very useful piece of equipment!

Galactose would be cool to measure but it's not absolutely necessary. There are definitely galactose colorometric kits out there that will allow us to use our spectrophotometer to measure galactose.

High yield batch-fed culture

To get high concentrations Kim et al. used a batch-fed bioreactor and in the paper they detail the specifics of how they initialize it but the most important part is probably this:

During the production phase, the glucose concentration was maintained below 1 g/l and the galactose concentration at approximately 15 g/l. The temperature and pH were maintained at 30°C and 5.0 – 5.5, respectively, throughout the bioreactor operation. -- Kim2015