SBB10AssayTeam1-Notes

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General

Potential Timeline

Day 1: make master mix for 40 constructs, 2 arabinose, 2 non-arabinose for each construct. and transform by heat shock, grow overnight.

  • JH - when are you putting the arabinose? Picking step, or transformation? What if the part is toxic (you can't tell why transformation failed?)
  • JK - the protocol we found said to combine the arabinose with the LB media after we pick the colonies.
  • JK - we need to make enough master mix to have controls as well
  • AL- check inventory for toxicity for the master mix for each batch.

Day 2: pick colony and grow to saturation with LB media.
Day 3: make arabinose and non-arabinose samples out of saturated bacteria mixture, apply antibiotics (K and C), grow.
Day 4: run TECAN with cell aliquot.
Day 2-5: repeat Day 1-4 for the rest of the constructs.

PowerPoint

http://docs.google.com/present/edit?id=0AYl_c3UjUjSqZGZ3Z2o5eHpfMjdmandiZzNjZg&hl=en

Parts list

Media:140L_parts1_032910.xls

Vector

Toxicity

Toxicity Protocol

Controls: DH10B (blank), and pBca????-Bca1144 (plasmid effects.)
Parts: 2 arabinose, 2 non-arabinose

  • Take 2 tubes of 280ul of cell and add 60ul KCM and 100ul water to each.
  • Add 20ul of the KCM/water/cell solution into each construct.
  • Transform (heat shock, etc.)
  • Grow plates overnight.
  • Pick 6 colonies from each sample.
  • Grow to saturation in 96 well blocks with 400 uL LB media in each well. (16 parts total per well)
  • Then from each of the 6 unique liquid cultures, make 3 arabinose samples and 3 non-arabinose samples
  • Add 50 uL of LB media or LB Media+100ug/mL arabinose See note below per well in 384 well plate.

You should make cocktails of LB with whatever antibiotics or arabinose you need. Kanamycin and Chloramphenicol are stored at 25mg/mL. Ampicillin is stored at 100mg/mL. Arabinose is stored at 100mg/mL. At these concentrations, they are 1000x. You want to start with the same batch of LB for all samples in your plate. Calculate what total volume of each mixture of additives you'll need, and make up the mixtures in Eppendorf plates, mix well, then transfer the aliquots to the 384-well plates.
So assuming everything is at 1000X concentrations, we need to dilute everytion to 1x in a 50ul LB media. This means that everything needs to be 1000x more dilute than it presently is. In order words we need just need to add a concentration that is 1000 times less than 50ul which is: 0.05ul of each in the 50 ul lb or 0.4ul of each in the 400ul lb
Lastly, for quantities of everything, we need (1/1000*400ul*19) or 7.6 ul of arabanose. We need (400ul*19) or 7.6ml (7600ul) of LB. All of our parts are CA (JH - really?! makes our life easier.) so we need (1/1000*400ul*19) or 7.6 ul of both Ampicillin and Chloramphenicol. 

So in short: 
Chloramphenicol 6.8ul
Ampicillin 7.2ul
arabanose 7.6ul
LB 7.6ml
Plasmids DH10B (no antibiotics), and pBca????-Bca1144.
  • We made 2 tubes of LB. One 850ul tube with 0.85ul of arabinose and one with no added arabanose. We did not need to add antibiotics as it was premixed.
  • We added 50ul of LB (no arabinose) to # wells
  • We added 50ul of LB with arabinose to # different wells
  • In each of the wells, we added 1ul of the appropriate vector
  • We put our sample in the TECAN for 36 time points, 10 minutes between each.

Reagent inventory

Toxicity

http://spreadsheets.google.com/ccc?key=0AsHu1Wyunvu3dHhoSGxrVVFCc0hJM2xZNmNEUFUtUVE&hl=en
Notes:

  • JH - not strictly necessary but we might confirm that both tag and untagged variants that are otherwise identical are needed.
  • JH - Only 8 units are specified for transformation. Is this enough, because Template:SBB-Protocols_Micro1 states to use 70uL per ligation. If we do the math, (200+50+30)/70 = 4, or 3 to allow for inexact volumes (alternatively, you could bump up the amount used by a bit, or bump down the amount to put in ligation to 65 uL, etc). If you read carefully, you will see 2YT (100) is needed for EACH ligation/transformation. Putting it as part of MM will not work (well).

[math]\displaystyle{ Insert formula here }[/math]

Transformation

Competent cells are stored as 280uL aliquots in the -80 freezer as a communal stock.
Volumes below have been corrected.

  1. Thaw 1 tubes of 10 uL aliquot of cells on ice
  2. Add 2.5 uL of water to each tube
  3. Add 1.5 uL of KCM salts to each tube
  4. Add 0.5 ul of the constructs to 10 uL of the cell cocktail. Pipette up and down gently to mix
  5. Let sit on ice for 10 min
  6. Heat shock for 2 min at 42
  7. Put back on ice for 1 min
  8. For ampicillin selection, you can plate immediately, otherwise:
  9. Add 100uL of 2YT, let shake in the 37 degree incubator for 40 min
  10. Plate on selective antibiotics, let incubate overnight

TECAN Safire II operation protocol (96-well)

A TECAN Safire II machine at Architecture et Fonction des Macromolécules Biologiques

Running a TECAN analysis:
The procedure below only works for black 96-well flat bottom plates.

  1. Turn TECAN power on (wait for light to stop flashing)
  2. Open XFluor4 Safire II XLS spreadsheet
  3. Load plate:
    1. Have 96-well plate with media+cells
    2. Goto XFluorSafireII menu > movements > out
    3. Load plate
    4. Goto XFluorSafireII menu > movements > in
  4. Load the program to run:
    1. Select Multi Labeling Kinetic
    2. Load multi labeling kinetic parameter > "\iGEM 2007\My Documents\Weston\gabe's experimental folder\kinteticmodified_no gfp reading.mps"
    3. Click "Run"
  5. Wait for data collection. Operation can only be cancelled when machine is performing measurements.

Questions

  • What are the parts to analyze? Basically everything on the parts list. Expect to use 3-4 plates
  • What plasmid are we using? Antibiotic selection markers? A R6K - Kanamycin - Chloramphenicol plasmid with various selectable antibiotic markers (see "vectors" section).
  • Number of replicates to do? 2 is sufficient.

Powerpoint comments by JCA

  • If we have R6K plasmid, we need DH10B with pir+. ...right? AFAIK MC1061 pir+ is the default pir strain
  • All samples can be done at once - high throughput ftw.

Expression

  • Plate washer: improves data quality and reproducibility
  • rbs-prepro - periplasmic - deoxycholic acid
  • rbs - cytoplasmic - "Bugbuster"

Questions

  • Theoretical:
  1. Tags to use? (i.e. Myc, 6His, etc). Everything. The ones I know of: Myc, His, HA, FLAG
  2. Antibodies on plate? We will likely be doing a sandwich ELISA, so yes.
  3. How to make this quantitative? How much protein DO you have? Most likely reporting OD should be sufficient. Ask JCA for more details.
  • Experimental:
  1. Available primary antibodies? Concentrations? JCA - we may have 2nd antibody covalently attached to HRP
  2. Is the first antibody bound to the plate already? Do as protocol indicates. The first blocking step is the crucial one.
  3. Experimentally "sound" concentration BSA / time to block?
  4. Detection - ABTS: how much, how long?

Powerpoint comments - JCA

  • Don't need 2 batches.
  • All samples can be done at once - high throughput ftw.
  • Use PBS (+ Tween) instead of CB1 or stupid proprietary buffers. This will depend on the kit though.
  • We will talk about using positive controls ... they probably have single-tag purified proteins, and protein fusions can create "dual tags", though not identical to our dual tag configuration.

General protocol

Sample Prep

  • Combine cell aliquots, KCM, and water
  • Transform (heat shock, etc.)
  • Grow plates overnight
  • Pick colonies from each sample
  • Lyse cells: Add correct reagent to blocks, shake at 37C for 1 hour

Reagent inventory

ELISA

http://spreadsheets.google.com/ccc?key=0AvbXytCFRyFCdFdnbW15cENsQll0ZUJpYXFhbWRrX3c&hl=en
Notes:

  • JH - Please put explanations of color coding on Sheet 1 somewhere (comments, or here)

ELISA

JH - I think I finally got all the controls figured out for ELISA after a bit of pondering. Check the powerpoint.
Possible protocol (ABTS concentration): http://www.southernbiotech.com/techbul/5300-05.pdf
Another protocol (with some notes on concentration of target protein): http://www.abcam.com/index.html?pageconfig=resource&rid=11388

  1. JH - And ... how do we know the concentration of protein if we haven't done the ELISA? Unless I'm reading this wrong, it's a Catch-22.
  1. JCA - CB1 is 99% likely to just be PBS. Use PBS
  2. Adding some Tween (detergent) to PBS may improve results.
  • Add appropriate antibody to each dilution, to a concentration of .1-10 ug/mL (maybe one dilution of CB1 per type of tag?)
  • Pipette solution into the plates, between 50 and 300 uL per well
  • Incubate 3-24 hrs at room T, protect from light, minimize evaporation by covering the top of each well
  • After incubation, dump coating solution out of wells (the antibodies are now coating the bottom of each well)
  • Wash 2-4 times with ELISA wash buffer
  • Add 300-400 uL block buffer 1 to "minimize nonspecific adsorbtion and protect the antibodies from harsh external conditions, while blocking uncoated regions of the well"
  1. JCA - just use BSA or milk.
  • Incubate as before (3-24 hr, room T, no light etc)
  • Aspirate block buffer
  • Add cell lysate into wells on plate
  • Incubate
  • Wash off unbound lysate and rinse with PBS
  • Block with BSA
  1. JCA - this 2nd block is not critical, but can be helpful. Mix the block in dilute lysate.
  • Wash with PBS
  • Add anti (something) antibodies
  • Incubate
  • Wash with PBS
  • Add 2nd antibody conjugated with streptavidin/HRP, incubate
  • Wash with PBS
  • Add detection solution, incubate for a specific time
  • Add stopping solution
  • Read absorbance with ELISA plate reader
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