We know you are excited to solve the puzzle challenge
However, for the best playing experience
we would suggest switching to a device with larger screen size (at least an iPad viewing in landscape)
See you there! :)
Experimental design tactics - PLAN
Before you try to design a good experiment, you need to first understand the technique you are using and what you are expecting. Click here to review your understanding of SPR or ask the demonstrator.
The best approach against an unknown system is to "try and adapt", rather than sticking to a particular method. This guide will help you utilise your understanding of SPR to systematically design an efficient experiment via the Plan-Test-Reflect Cycle framework.
1. Plan Phase a) Backward derivation method
Start thinking from the end point will help you derive what you experimental steps are important or unnecessary to prove your hypothesis by your variable.
- What is your hypothesis?
- What independent and dependent variables can we measure to provide evidence for that?
- What technique(s) can help you directly/indirectly measure the variable?
- Which technique gives you the best precision and least false positives and negatives?
- List out all steps require to derive the variable from raw data output by the technique (e.g. graph plotting, trend extrapolation, calculations).
b) General experimental design considerations
Every experimental dataset accepted as scientifically sound by the scientific community share a common sets of characteristics. Integrate them into your experimental steps.
- 6 repeats are required for results to be 95% confidence, in addition to calculating the standard deviaiton of the technique.
- 3 independent repeats are required per data point to identify possible outliers. You will not know which point is the outlier if you only have 2 points.
- The absolute and relative error affects the range of values you can sample your data from. In particular, absolute error gives you the lowest sampling limit.
- Have you remove the background? This is particularly important if background is not a constant.
- What variables could alter the results such that you need to control for?
c) SPR specific considerations
- What range of Kd can you expect in strong and weak binding? Have your experiment accounted for the broad range of possible answers?
- Have you use an Association Time of sufficient length for the system to reach equilibrium binding? How do we know if equilibrium binding is reached in SPR?
- What trend are you expecting for Langmuir Isotherm? Thus how many data point do you need to sufficiently describe the trend (e.g. shape of curve)?
Experimental design tactics - TEST
2. Test Phase
You can now translate all experimental design considerations you just derived into specific experimental steps taken to find the results.
What is most important when tackling an unknown system is to have the metacognition to accept that you will NEVER get the design right all at once. The most important point, again, is to iterate your Plan-Test-Reflect Cycle as quick as possible rather than worrying that you might miss out on any other considerations! This is because you can learn much faster about what is missing through testing out your plans right away.
So quickly try out your plan! When you finish, we can start to reflect on how to improve the design in the Reflect phase. Do note down your Efficiency Points and Steps taken so we can analyse them later.
Experimental design tactics - REFLECT
3. Reflect Phase
If you have successfully obtained the correct answer but not with the right Efficiency Points and Steps, troubleshoot with the hints below then quickly start the Plan-Test-Reflect Cycle again! If you have not obtain the right answer or still not able to achieve the VICTORY CONDITIONS, ask the demonstrator for help.
Efficiency Points
- Using your knowledge that the SENSORGRAM binding curve follow a hyperbolic model, try seeding a small value and predict the resulting trajectory where equilibrium binding would be reached, rather than randomly input a large value. In a real experiment, you would not have the time or resource to waste using the latter strategy.
Steps
- If you have the problem of needing to find a new equilibrium time for every new Concentration input, try finding a universal association time to use. What Concentration requires the longest time to reach equilibrium? Using this time length would cover for all cases.
- Did you do sufficient repeats to identify outlier and standard deviation of the technique?
- Did you use a broad sampling strategy to account for all possible range of value Kd could take?
- How do you confirm saturation has been reached?
- How did you find where the dynamic range of Langmuir Isotherm lies? Knowing that Langmuir Isotherm follow a hyperbolic model, it would be wise to start gathering points from lower concentration upwards, you might still be sampling points in the saturation range if you do the reverse.
- Think again about the final end point calculations you took to derive the answer. From there, are there unnecessary steps you performed irrelevant to finding the answer? Can you combine some of the steps?
Experimental design tactics - ANALYSE
4. Analyse Phase
This list shows the common considerations to be mindful of when working with resulting data.
Data Display
- UNITS!!!
- Graph have title, units on both axes, error bars and not including outlier in trendline
- Be quantitative in represeting data
- Always perform statistical analysis
- Significant figures up to the least sensitive technique use
- Show n-value
- Represent data in format with highest information density possible (e.g. table format, describe relationship using equation)
- If a variable occupy a broad magnitude range, plot them in a log scale
Data Analysis
- When analysing, two data sets are comparable only if one factor differ, all else being equal
- Be concise and selective when explaining
- State definitions and assumptions
- State advantages and limitations of technique
- State what variables can influence the result when affected, thus needed to be control for
- State what further experiments or other techniques use would you do that can expect to support conclusion
- State what the data does not tell you, and what further experiments would you need to validate the hypothesis
- Never over conclude data; There is a scientific difference between 'proves', 'suggests' and 'is consistent with'
5. Final Note
Remember, the Plan-Test-Reflect Cycle is universally applicable to all experiments, not just SPR. Try using it next time to plan your experiments!
Confirm Home
Please confirm if you want to finish the experiment for the day. After you click, you will have {{view.experiment.daysLeft}} full day(s) left to perform your experiment.
Confirm Clear
Please confirm that you want to clear the SENSORGRAM. The data in the RESULTS TABLE will not be clear. It is advised that you take a screenshot of the SENSORGRAM before clearing.
Confirm Restart Game
Are you sure you wish to restart your experiment? The same pair of ligand-receptor will be retain but all data in the table previously generated will be clear.
If you wish to keep the data before restart, export it as excel by clicking on the menu button at the top-right corner of RESULTS TABLE header.
Confirm New Game
Are you sure you wish to replay? A new pair of ligand-receptor will be generated and all data in the table previously generated will be clear.
If you wish to keep the data before restart, export it as excel by clicking on the menu button at the top-right corner of RESULTS TABLE header.
Incomplete experiment detected
We have detected an auto-saved incomplete experiment before you left the simulator. Do wish to continue with the auto-saved experiment or reload a new pair of ligand-receptor?
Experimental Design Strategy Puzzle (SPR)
Challenge Statement
So you think you have mastered experimental design strategy? We challenge you to crack this puzzle to see how good you are.
Victory conditions
+ Achieve the correct Kd, kon, koff within the days allowed
+ Using the Optimum Steps, excluding outliers
+ Without losing any Efficiency Points
+ Making at least {{view.experiment.comboStreakMax}} experimental design considerations
Before you begin the game
You should be comfortable with how to obtain Kd, koff and kon using Surface Plasmon Resonance (SPR) technique before attempting to design a good experiment to complete the challenge. Please also read the HELP section in CONTROL PANEL to understand game mechanics.
Let's begin by selecting a suitable playing mode. If you are new to experimental design strategy, Training mode will teach you how to beat the challenge with the Strategies & Tactics and Console. Otherwise, you can try to beat the Challenge straight away!
Note: Please use Google Chrome browser to ensure full functionality of software.
Game Mechanics
If you need to quickly recall basic SPR Theory, click here.
1. Basics
- Input desired Free Ligand Concentration and Association Time
- Click START to begin ligand-receptor association phase
- Click WASH to begin ligand-receptor disassociation phase
- Click BACKGROUND to remove it from future readings
- Check your answer in the CHECK ANSWER panel to complete the game
2. Essential tips to completing the game
- Absolute and relative errors are built into the game.
- The probability of outputting results with high standard deviation increases after 17.00, and even further at 21.00.
- A maximum of 10 curves can be display on SENSORGRAM before CLEAR is required.
- You can drag an area on SENSORGRAM to zoom into it, double click to zoom out.
- Every RUN-WASH cycle uses up 30 minutes per day regardless of Association Time input.
- You can download your data into an Excel file by clicking the menu-button in the top-right corner of RESULTS TABLE.
- You can let your the RESULTS TABLE show display data in order of latest trial by clicking on 'trial' column to sort it
- Efficiency points deduction are calculated by how far off your Association Time input is from the time needed to exactly reach equilibrium (with 5 seconds buffer)
3. SPR assumptions
- No baseline drift in background
- Absolute error of SPR machine is +/-4 nM
- Both ligand and receptor are proteins
- Binding stoichiometry is a 1:1 interaction.
- No non-specific binding
Your answers are all in the right range! However, it appears that you have not achieve them within the VICTORY CONDITIONS. :(
To help you achieve the VICTORY CONDITIONS, you can download your data from the RESULTS TABLE and reflect upon how to improve your Efficiency or Steps Count with the demonstrator. Afterwards, click RESTART GAME and switch to TRAINING mode to access the Strategies & Tactics section and helpful Console tips to improve your method.
Good luck! You are not far from winning the game!
Congratulations, your answers, Steps Count and Efficiency Rating are all in the right range. Check with the demonstrator to confirm result is correct! :)
If you want to replay again with a new pair of ligand-receptor, click .
If you like this game, please provide feedback or challenge your friends who might be interested!
Remember that these Strategies & Tactics you learnt today are generally adaptable to all experimental techniques!
You can download a PDF summary of the Strategies & Tactics with additional notes on identifying flaws in a technique here.
Try applying them now to another Enzyme Kinetics simulator here. Good luck!
> Console:
[Lunch]
12.00-13.00: Had lunch at Biochemistry Cafe with friends
13.00-13.30: Began new experiment after lunch
> Console:[End of official working hour]
You are feeling tired. Output will now have an intermediate increase probability of getting higher standard deviation. You can continue your experiment now or resume tomorrow by clicking HOME.
> Console:
[Dinner]
17.00-18.00: Had steak & ale pie at The Eagle & Child with friends
18.00-18.30: Began new experiment after the dinner
> Console:[Exhaustion warning]
You are feeling exhausted. Output will now have a high increase probability of getting higher standard deviation. You can continue your experiment now or resume tomorrow by clicking HOME.
> Console:
[Supper]
21.00-22.00: Had mars bars alone in the lab, poor you.
22.00-22.30: Began new experiment after supper
> Console:[Security guard warning]
The security guard is closing the Biochemistry building.
Unfortunately, you will have to end the experiment now and go HOME.
> Console:
[End of a day @ {{view.experiment.endOfExperimentTime | timeFilter}}]
Automatic lab clean-up by CleanBots. . . Clean-up completed. (wish this was real)
See you tomorrow!
> Console:
[Start of a new day @ 9.00]
Experiment automatically setup. . . Setup completed @ 9.30. Ready to begin.
> Console:
[Hint: How to begin]
Please input values for Free Ligand Concentration and Association Time in CONTROL PANEL to begin. Click START and then WASH to begin the association and disassociation phase respectively.
> Console:[Plan first!]
Wait! Before you start, have you plan your experimental strategy? Never start an experiment without a plan, otherwise Steps and Efficiency Points could be wasted. If you are not sure where to start, click the Strategies & Tactics section for help!
> Console:[Hint: Remove background]
Before trying any other combinations, you should set the background value to remove it from subsequent experiment. The option to set will appear after you START and WASH the first trial with free ligand concentration = 0 M with any Association Time input. If you haven't set background after the first round, scroll down and click RESTART GAME.
> Console:[Hint: Find the right association time to use first]
Two possible scenarios:
i. This Association Time used might be insufficiently long to reach equilibrium binding
ii. Although equilibrium binding is reached in this Association Time, this Association Time will not universally give equilibrium binding when applied on lower Association Time which you might need to sample later.
> Console:[Hint: Absolute error]
Be careful of the output results in this Free Ligand Concentration range since the absolute error of SPR could take up a high proportion of it.
> Console:[Invalid Free Ligand Concentration input]
Free Ligand Concentration can only intake a number equal or above {{view.output.minimum_fLC_input}}.
> Console:[Invalid Association Time input]
Association Time input can only intake a number between 1 to 900.
> Console:[Maximum no. of plot on sensorgram reached]
SENSORGRAM can only display 10 curves at once. Please CLEAR your SENSORGRAM before you continue. The data in your RESULTS TABLE will not be cleared.
> Console:[Trial {{view.experiment.steps}} - steps status]
Your steps count is above the optimum steps. In the lab, this will waste precious time and resources. So finish off your experiment, download your data from RESULTS TABLE, and using the framework in Strategies & Tactics section as a guide, reflect on the steps you took and think of how to reduce your steps count.
> Console:[Consideration made: outlier]
Three replicates made per point will allow you to identify outliers. Having two is insufficient to distinguish which one is the outlier.
Six replicates made per point will allow you to calculate the standard deviation of the technique for both the association and disassociation phase. The answer of this trial will also be 95% confidence.
Considerations Made +1!
> Console:[Consideration made: sufficient points to describe trend]
You have sampled at least six different Free Ligand Concentration input (excluding background setting). Number of different points to sample is determined by the expected shape of line since there must be sufficient points to describe the trend (e.g. Hyperbolic model in Langmuir Isotherm)
You've found the universal Association Time that is of sufficient length to ensure equilibrium is reached in any Free Ligand Concentration input. The lower the free ligand concentration, the longer time it takes to reach equilibrium. So finding association time for your lowest magnitude you would sample gives an Association Time you can use universally at all higher concentration.
Saturation is confirmed when a second higher Free Ligand Concentration input is used and the resulting SENSORGRAM output shows an overlap of the curve.
Although the binding constant of the ligand-receptor pair is unknown, we know that in Biochemistry, the Kd usually lies betwen mM (weak binding) to nM (strong binding) range. Therefore, only by using a broad sampling strategy, rather than incrementing at a particular magnitude can you efficiently find the saturation point and dynamic range of Langmuir Isotherm. (In this case, sample at mM and nM level)
Considerations Made +1!
> Console:[Consideration made: remove background]
Background had been set at {{view.backgroundSet}} {{view.backgroundUnitsSet}}.
Removal of background is important to ensure any change is the result of ligand binding, not receptor change, especially since background is not always constant in SPR.
Considerations Made +1!
{{view.experiment.inefficiency_display}} Effiency Points lost!
You are allowed a buffer of 5 extra seconds after equilibrium binding is reached before efficiency points get deducted
0 Effiency Point lost, keep it up!
> Console:[Experiment Ended]
You ran out of time to finish the experiment. To try again without changing the system, click RESTART GAME.
RESULTS TABLE
YOUR EXPERIMENT STATUS
Days Left
{{view.experiment.daysLeft}}
Time of Day
{{view.experiment.timeOfDay | timeFilter}}
Efficiency Points
{{view.experiment.efficiencyRating}}
Steps Taken
{{view.experiment.steps}}
CONTROL PANEL
Free Ligand Concentration
Association Time
seconds
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