20.109(S14):Protein-level and wrap-up analysis (Day7): Difference between revisions

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==Introduction==
==Introduction==


Last time, you learned the solar conversion efficiency of the DSSC that you personally manufactured, and started to get a sense of how your condition compared to others. (Note: In a previous year, typical efficiencies ranged from about 1.5-5.5%.) Today you will finish interpreting and writing up this class-wide data.
Today we’ll complete the final piece of analysis for module 3, namely quantifying the collagen protein content in samples cultured under different conditions. You should spend the remainder of your time today finishing your module 3 report and preparing for your research proposal presentation.


To calculate DSSC efficiency, you also measured the open circuit voltage (Voc) and short circuit current (Isc). Separately, you quantified the DSSC fill factor, which essentially measures how well the device performs compared to its ideal capacity. You can learn much more about these measures at the [pveducation.org Photovoltaic Education Network website], particularly the Solar Cell Operation section. Some brief context about Voc and Isc is also included below, modified from the 20.309 Electronics Primer that one of your instructors helped write.
==Protocols==
 
===Part 1: ELISA analysis===
 
The analysis of protein concentration that you perform today will be similar to the titration curve analysis that you did in Module 2.


[[Image:309_epd_resistor-fully-labeled.png|thumb|225px|right|'''Figure 1: Resistor with terminal parameters defined.''' The voltage across and current through the element are shown.]]
#Open the text file containing your raw data in Excel, and save it as an Excel file.
First, recall that current (i) reflects the flow rate of charge carriers such as electrons, while voltage (v) reflects the electrical potential difference between two points. The higher the voltage, the greater the current flow through a circuit element such as a resistor, all else being equal. Finally, note that resistance reflects the tendency of an element to draw current and also its tendency to dissipate energy. A linear resistor obeys Ohm’s law, <math>v = iR</math>, at any instant in time.
#Label the columns to reflect your samples. It may be easiest to visually separate samples (in the first few rows of the sheet) and standards (in the next few rows), since they will undergo partly different manipulations. You may also want to work on two separate worksheets, one for each collagen type.
#Average your replicate values for both standards and experimental samples.
#Now calculate the average of your blank samples, then subtract this background value from each of your raw averages. (So far your column headings might look like: REP1, REP2, AVE, AVE-SUB.)
#You will use your standard readings to make a calibration curve. Plot the absorbance readings for the standards (on the ''x''-axis) vs. the known concentration of collagen added (on the ''y''-axis). Just type in the first concentration, and divide by two down the column.
#Click in the chart area, then choose menu ''Chart'' &rarr; ''Add Trendline''. Click on the ''Options'' Tab, and choose to display both the equation and the R<sup>2</sup> value on the chart.
#Delete data points that seem to be outside the linear range of the assay (just delete the AVE-SUB value, not the raw data!), until you get a reasonable R<sup>2</sup> value for your line, i.e., one that is close to 1. The equation should update in real-time as you delete data.
#Now that you have the slope and intercept of the line, you can feed this information back into the absorbance values for your experimental samples, and calculate the actual protein concentrations. If you are unsure of how to proceed, ask your peers or instructors. The $ symbol in Excel is useful here for efficient calculations, in case you want to compare the results for multiple slope/intercept values.
#Your results will most likely be closer to ng/mL than &mu;g/mL, so go ahead and convert them.  
#Finally, address the following in your report:
#*which samples had a measurable amount of collagen I? collagen II?
#*for samples with both values in range of the assay, what was the collagen II:I ratio?
#*how do these results compare to those at the transcript level? what factors might cause any differences that you see?


Two limiting cases of the resistor element are the open circuit and the short circuit (Fig. 2). Infinite resistance means that no current passes through that element; this is also called an open circuit across said element and is equivalent to an unconnected port. Zero resistance means no potential difference appears across the element; this is also called a short circuit across that element and is equivalent to an ideal wire. In a device such as your solar cell, the values Voc and Isc are the maximum voltage and current that the device can attain, and their product is the ideal maximum power available. In turn, a high fill factor indicates low energy dissipation (through resistance internal to the device) and near ideal operation.
===Part 2: Complete report===


[[Image:309_epd_open-and-short-circuits.png|thumb|550px|center|'''Figure 2: Open and short circuits.''' In the open circuit (left), the maximum potential difference exists across the resistor, and <math>V_{AB} = V</math>. In the short circuit (right), the potential is the same everywhere because a wire connects the two resistor terminals, and <math>V_{AB} = 0</math>.]]
Your Module 3 report is due before you leave lab today. Please print a hardcopy of the report (double-sided, please!) in addition to emailing it in to the 20109.submit address.


==Protocols==
===Part 3: Prepare for presentation===


===Part 1: Complete mini-report===
Next time you will present your research proposals to the rest of the class. Now is a great opportunity to get feedback from your peers and/or the teaching faculty. If you did not complete your cross-group research discussion last time, remember to do so today.


Working with your partner, write a short report describing the Module 3 work according to the [[20.109%28F13%29:_Biomaterial_engineering_report | linked specifications]].
==For next time==


Please print a hardcopy of the report (double-sided, please!) before you leave in addition to submitting on Stellar. You may print just the text if the figures exceed the printer memory capacity.
Prepare a 12 minute powerpoint talk that describes the research question you have identified, how you propose to study the question, and what you hope to learn. More detailed descriptions of the elements of the oral presentation can be found in the FNT assignments and the protocols associate with this Module as well as with the [[20.109(S13):Module 3 oral presentations| research proposal guidelines]]. When it is ready, please email your presentation to 20109 DOT submit AT gmail DOT com. '''Speaking order will be determined by the order that presentations are received.'''


===Part 2: Closing down the lab...===
On the day you present (see announcements on front page for when and where) your team should print out and bring <b>two copies</b> of your powerpoint slides. Black and white is fine and you can print more than one slide per page if you like (4-6 slides per page tends to be ideal for my note-taking). You should also write (and print out) your "talking points" in the comments box of each slide. These are speaking notes for your presentation, and should include short phrases to remind you of the key points to cover on each slide, as well as the transitions you've planned between them. For example, in a previous year's presentation one slide's talking points were: <br>


Some time during lab, find 10-15 minutes to complete your section-specific items on the clean-up/inventory document.
<i>As you can see from this image, taken from a review on hydrocarbon metabolism in marine bacteria, the alcanivorax species is the first to grow in population after an oil spill, and its growth correlates with a decrease in aliphatic hydrcarbons.
*After most alkanes have been degraded, the Cycloclaticus species blooms while aromatic hydrocarbon levels decrease
*One thing to note is that as soon as they have done their job, both species return to their normal population levels.
*One problem with using Alcanivorax and Cycloclasticus to clean oil spills, however is that they can only be found in specific locations
</i>
The next slide (transition statement) began:
<i>To remedy this, we decided to look into other bacteria into which we could move the hydrocarbon metabolic pathways</i>


==For next time==
You don't have to use complete sentences in your own talking points, but the above example should give you a sense of what content is expected.


Prepare a 12 minute powerpoint talk that describes the research question you have identified, how you propose to study the question, and what you hope to learn. More detailed descriptions of the elements of the oral presentation can be found in the FNT assignments and the protocols associated with this Module, as well as in the [[20.109(F13):Module 3 oral presentations| research proposal guidelines]]. '''When it is ready, please email your presentation to 20109 DOT submit AT gmail DOT com. Speaking order will be determined by the order that presentations are received.'''
'''''You will be graded on the integrated success of your presentation: concepts, slides, talking points, and presentation.'''''


==Navigation Links==
==Navigation Links==
Next Day: [http://openwetware.org/wiki/20.109%28F13%29:_Mod_3_Day_7_Oral_presentations Mod 3 Day 7: Research Proposal Presentations]
Next Day: [[20.109(S14):Student presentations (Day8)| Student presentations]]
Previous Day: [http://openwetware.org/wiki/20.109%28F13%29:_Mod_3_Day_5_Solar_cell_testing Mod 3 Day 5: Solar Cell Testing]
Previous Day: [[20.109(S14):Transcript-level analysis (Day6)| Transcript-level analysis]]

Revision as of 13:04, 17 January 2014


20.109(S14): Laboratory Fundamentals of Biological Engineering

Home        Schedule Spring 2014        Assignments       
Module 1        Module 2        Module 3              

Introduction

Today we’ll complete the final piece of analysis for module 3, namely quantifying the collagen protein content in samples cultured under different conditions. You should spend the remainder of your time today finishing your module 3 report and preparing for your research proposal presentation.

Protocols

Part 1: ELISA analysis

The analysis of protein concentration that you perform today will be similar to the titration curve analysis that you did in Module 2.

  1. Open the text file containing your raw data in Excel, and save it as an Excel file.
  2. Label the columns to reflect your samples. It may be easiest to visually separate samples (in the first few rows of the sheet) and standards (in the next few rows), since they will undergo partly different manipulations. You may also want to work on two separate worksheets, one for each collagen type.
  3. Average your replicate values for both standards and experimental samples.
  4. Now calculate the average of your blank samples, then subtract this background value from each of your raw averages. (So far your column headings might look like: REP1, REP2, AVE, AVE-SUB.)
  5. You will use your standard readings to make a calibration curve. Plot the absorbance readings for the standards (on the x-axis) vs. the known concentration of collagen added (on the y-axis). Just type in the first concentration, and divide by two down the column.
  6. Click in the chart area, then choose menu Chart → Add Trendline. Click on the Options Tab, and choose to display both the equation and the R2 value on the chart.
  7. Delete data points that seem to be outside the linear range of the assay (just delete the AVE-SUB value, not the raw data!), until you get a reasonable R2 value for your line, i.e., one that is close to 1. The equation should update in real-time as you delete data.
  8. Now that you have the slope and intercept of the line, you can feed this information back into the absorbance values for your experimental samples, and calculate the actual protein concentrations. If you are unsure of how to proceed, ask your peers or instructors. The $ symbol in Excel is useful here for efficient calculations, in case you want to compare the results for multiple slope/intercept values.
  9. Your results will most likely be closer to ng/mL than μg/mL, so go ahead and convert them.
  10. Finally, address the following in your report:
    • which samples had a measurable amount of collagen I? collagen II?
    • for samples with both values in range of the assay, what was the collagen II:I ratio?
    • how do these results compare to those at the transcript level? what factors might cause any differences that you see?

Part 2: Complete report

Your Module 3 report is due before you leave lab today. Please print a hardcopy of the report (double-sided, please!) in addition to emailing it in to the 20109.submit address.

Part 3: Prepare for presentation

Next time you will present your research proposals to the rest of the class. Now is a great opportunity to get feedback from your peers and/or the teaching faculty. If you did not complete your cross-group research discussion last time, remember to do so today.

For next time

Prepare a 12 minute powerpoint talk that describes the research question you have identified, how you propose to study the question, and what you hope to learn. More detailed descriptions of the elements of the oral presentation can be found in the FNT assignments and the protocols associate with this Module as well as with the research proposal guidelines. When it is ready, please email your presentation to 20109 DOT submit AT gmail DOT com. Speaking order will be determined by the order that presentations are received.

On the day you present (see announcements on front page for when and where) your team should print out and bring two copies of your powerpoint slides. Black and white is fine and you can print more than one slide per page if you like (4-6 slides per page tends to be ideal for my note-taking). You should also write (and print out) your "talking points" in the comments box of each slide. These are speaking notes for your presentation, and should include short phrases to remind you of the key points to cover on each slide, as well as the transitions you've planned between them. For example, in a previous year's presentation one slide's talking points were:

As you can see from this image, taken from a review on hydrocarbon metabolism in marine bacteria, the alcanivorax species is the first to grow in population after an oil spill, and its growth correlates with a decrease in aliphatic hydrcarbons.

  • After most alkanes have been degraded, the Cycloclaticus species blooms while aromatic hydrocarbon levels decrease
  • One thing to note is that as soon as they have done their job, both species return to their normal population levels.
  • One problem with using Alcanivorax and Cycloclasticus to clean oil spills, however is that they can only be found in specific locations

The next slide (transition statement) began: To remedy this, we decided to look into other bacteria into which we could move the hydrocarbon metabolic pathways

You don't have to use complete sentences in your own talking points, but the above example should give you a sense of what content is expected.

You will be graded on the integrated success of your presentation: concepts, slides, talking points, and presentation.

Navigation Links

Next Day: Student presentations Previous Day: Transcript-level analysis

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