Difference between revisions of "20.109(S21):M3D4"

Revision as of 15:19, 19 April 2021

20.109(S21): Laboratory Fundamentals of Biological Engineering

Spring 2021 schedule FYI Assignments Homework Communication | Accessibility

M1: Antibody engineering M2: Drug discovery M3: Protein engineering

Introduction

Protocols

As you consider sites that may alter calcium binding, keep the following in mind:
- When this module was first debuted, everyone mutated residues directly in the calcium binding loops, and very few groups saw dramatic changes in affinity or cooperativity of calmodulin with respect to calcium.
- In some years, class-wide results suggested that mutations in the first two binding loops were more likely to have an effect than mutations in the latter two binding loops.
- Some folks also targeted non-binding structural areas, but results were inconclusive.
- You may repeat or otherwise build upon prior results as long as you give your own reasoning.

Part 3: Primer design for mutagenesis

Primer design schematic for NEB Q5 Site Directed Mutagenesis.

It wouldn’t be very experimentally efficient to somehow pick out and modify a single residue on inverse pericam post-translationally. Instead, researchers genetically encode desired mutations, by making mutated copies of a plasmid that contains the inverse pericam DNA sequence. In addition to non-mutagenic amplification of a specific piece of DNA, synthetic primers can be used to incorporate desired mutations into the DNA. Primer design for site-directed mutagenesis is quite straightforward: the forward primer introduces a mutation into the coding strand. Both non-mutagenic and mutagenic amplification require cycles of DNA melting, annealing, and extension.

Remember from Day 1 that primers used in PCR amplification must meet several design criteria in order to ensure specificity and efficiency. Consider the following design guidelines for mutagenesis primers and think about how these differ from the guidelines for non-mutagenic amplification:

The desired mutation (1-2 bp) must be present in the middle of the forward primer.
The forward and reverse primers should 'face' away from the mutation and be 'back-to-back' when annealed to the template.
The primers should be 25-45 bp long.
A G/C content of > 40% is desired.
Both primers should terminate in at least one G or C base.
The melting temperature should exceed 78°C, according to:
- T_m = 81.5 + 0.41 (%GC) – 675/N - %mismatch
- where N is primer length, and the two percentages should be integers.

To demonstrate primer design, the illustration below uses S101L, which is an uninteresting mutation but is a straightforward teaching example.

Residue 101 of calmodulin is serine, encoded by the AGC codon. This is residue 379 with respect to the entire inverse pericam construct, and we can find it and some flanking code in the DNA sequence from Part 2:

361 (5') GAG GAA ATC CGA GAA GCA TTC CGT GTT TTT GAC AAG GAT GGG AAC GGC TAC ATC AGC GCT (3')

381 (5') GCT CAG TTA CGT CAC GTC ATG ACA AAC CTC GGG GAG AAG TTA ACA GAT GAA GAA GTT GAT (3')

To change from serine to leucine, one might choose TTA, TTG, or CTN (wherer N = T, A, G, or C). Because CTC requires only two mutations (rather than three as for the other options), we choose this codon.

Now we must keep >10 bp of sequence on each side in a way that meets all our requirements. To quickly find G/C content and see secondary structures, look at the IDT website. (Note that the T_m listed at this site is not one that is relevant for mutagenesis.)

Ultimately, your forward primer might look like the following, which has a T_m of almost 81°C, and a G/C content of ~58%.

5’ GG AAC GGC TAC ATC CTC GCT GCT CAG TTA CGT CAC G 3'

The reverse primer is the inverse complement of a sequence just preceding the forward primer in the IPC gene. The forward and reverse primers are set up back-to-back.

Lucky for us, NEB has a tool that can design our mutagenic primers.

Go to the NEBaseChanger site and click 'Please enter a new sequence to begin.'
- A new window will open. Copy and paste the wild-type IPC sequence.
Confirm that the 'Substitution' option is selected.
Highlight the basepairs you want to mutate using by scrolling through the sequence, or you can search the sequence by typing the basepairs into the 'Find' box.
Type the new DNA sequence (the basepair(s) you want your forward mutagenic primer to incorporate into the IPC sequence) in the 'Desired Sequence' box.
- Under the Result header, a diagram showing where your primers will anneal is provided.
- Under the Required Primers header, the sequences for your forward primer and reverse primer are shown with the characteristics for each.
Screen capture the information provided in the Result and Required Primers sections.
- Embed the images in your notebook.
- Print the screen capture and submit it to the teaching faculty before you leave today. In addition, record your primer sequences in the table on the Discussion page.
- It is very important that you submit your primer sequences before you leave! The teaching faculty will order your primers from IDT DNA tonight to ensure they arrive by your next class.
Use the guidelines above to examine the mutagenesis primers designed by NEBaseChanger. Include your thoughts in your notebook.
- Do NOT alter the primers provided by NEB.

Part 2: Primer preparation

While you were away the sequences for the mutagenic primers you designed were submitted to Integrated DNA Technologies (IDT). IDT synthesized the primers then lyophilized (dried) them to a powder. Follow the steps below to resuspend your primers.

Centrifuge the tubes containing your lyophilized primers for 1 min.
Calculate the amount of water needed for each primer (forward and reverse, separately) to give a concentration of 100 μM.
Resuspend each primer stock in the appropriate volume of sterile water, vortex, and centrifuge.
Now prepare a dilution from your archival stock. Prepare 100 μL of a solution that has both the forward and reverse primers, each primer at 10 μM.
- Try the calculation on your own first. If you get stuck, ask the teaching faculty for help.
- Be sure to change tips between primers!
Return the rest of your primer stocks, plus your primer specification sheets, to the front bench.

Part 3: Site-directed mutagenesis

Schematic of NEB Q5 Site Directed Mutagenesis Kit procedure modified from NEB manual.

We will be using the Q5 Site Directed Mutagenesis Kit from NEB to perform your site-directed mutagenesis reactions. Each group will set up one reaction, for your X#Z mutation. Meanwhile, the teaching faculty will set up a single positive control reaction, to ensure that all the reagents are working properly. You should work quickly but carefully, and keep your tube in a chilled container at all times. Please return shared reagents to the ice bucket(s) from which you took them as soon as you are done with each one.

Get a PCR tube and label the top with your mutation and lab section (write small!).
Add 10.25 μL of nuclease-free water.
Add 1.25 μL of your mutagenesis primer mix (each primer should be at a concentration of 10 μM).
Add 1 μL of IPC template DNA (concentration of 25 ng/μL).
Lastly, use a filter tip to add 12.5 μL of Q5 Hot Start High-Fidelity 2X Master Mix - containing buffer, dNTPs, and polymerase - to your tube.
Once all groups are ready, we will begin the thermocycler, under the following conditions:

Segment	Cycles	Temperature	Time
Initial denaturation	1	98 °C	30 s
Amplification	25	98 °C	10 s
		55 °C	30 s
		72 °C	2 min
Final extension	1	72 °C	2 min
Hold	1	4 °C	indefinite

After the cycling is completed, the teaching faculty will complete the KLD reaction (which stands for "kinase, ligase, DnpI") using 1 μL of your amplification product, 5 μL 2X KLD Reaction Buffer, 1 μL KLD Enzyme Mix, and 3 μL nuclease-free water. The reactions will be incubated for 5 min at room temperature.

The teaching faculty will then use 5 μL of the KLD reaction product to complete a transformation into an E. coli strain (NEB 5α cells of genotype fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17) that will amplify the plasmid such that you are able to confirm the appropriate mutation was incorporated. The transformation procedure will be as follows:

Add 5 μL of KLD mix to 50 μL of chemically-competent NEB 5α.
Incubate on ice for 30 min.
Heat shock at 42 °C for 30 s.
Incubate on ice for 5 min.
Add 950 μL SOC and gently shake at 37 °C for 1 h.
Spread 50 μL onto LB+Amp plate and incubate overnight at 37 °C.

Reagent list

Q5 Site Directed Mutagenesis Kit from NEB
- Q5 Hot Start High-Fidelity 2X Master Mix
  - Propriety mix of Q5 Hot Start High-Fidelity DNA Polymerase, buffer, dNTPs, and Mg²⁺.
- 2X KLD Reaction Buffer
- 10X KLD Enzyme Mix
  - Proprietary mix of kinase, ligase, and DpnI enzymes.
The SOC medium contains 2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose.
LB+Amp plates
- The Luria-Bertani (LB) broth contains 1% tryptone, 0.5% yeast extract, and 1% NaCl
- Plates prepared by adding 1.5% agar and 100 μg/mL ampicillin to LB

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Difference between revisions of "20.109(S21):M3D4"

Revision as of 15:19, 19 April 2021

Contents

Introduction

Protocols

Part 3: Primer design for mutagenesis

Part 2: Primer preparation

Part 3: Site-directed mutagenesis

Reagent list

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@@ Line 5: / Line 5: @@
 ==Introduction==
-As evidenced by Nagai’s work, wild-type inverse pericam is not toxic to BL21(DE3)pLysS cells. Although it is unlikely that your point mutation will dramatically change this fact, in general a novel protein may turn out to be toxic. If this is the case, only very small amounts of protein are produced before the bacteria die. Keep in mind that overexpressing a single protein may come at the expense of producing proteins needed for survival, and will most likely cause cell death eventually; however, toxic proteins hasten this demise. Aberrant toxicity can sometimes be alleviated by reducing the culture temperature (e.g., to 30 °C).
+==Protocols==
-Based on its fluorescence activity, wild-type inverse pericam allows proper folding of (cp)EYFP, and based on its response to calcium, it also allows calmodulin to fold. One problem you may encounter is that your mutant proteins will no longer fold correctly. Since you made mutations in the calcium sensor part of IPC, rather than the fluorescent part, it is unlikely that your protein will destroy EYFP fluorescence. However, a common problem with misfolded proteins is the formation of insoluble aggregates, due for instance to improperly exposed hydrophobic surfaces. Proteins can be purified from these aggregates – called inclusion bodies – but the process is more labor-intensive than for soluble proteins. (The proteins must be extracted under more harsh conditions than you will use next time, then purified under denaturing conditions, before finally attempting to renature the proteins.) Inclusion bodies sometimes form simply due to very high expression of the protein of interest, causing it to pass its solubility limit. This outcome can be prevented by lowering the culture temperature, the induction duration, the amount of IPTG, or the growth phase of the bacteria.
+#As you consider sites that may alter calcium binding, keep the following in mind:
+#*When this module was first debuted, everyone mutated residues directly in the calcium binding loops, and very few groups saw dramatic changes in affinity or cooperativity of calmodulin with respect to calcium.
+#*In some years, class-wide results suggested that mutations in the first two binding loops were more likely to have an effect than mutations in the latter two binding loops.
+#*Some folks also targeted non-binding structural areas, but results were inconclusive.
+#*You may repeat or otherwise build upon prior results as long as you give your own reasoning.
-One final point to keep in mind is that not all proteins can be produced in bacteria. Eukaryotic proteins that require post-translational modifications (such as glycosylation) for activity require eukaryotic hosts (such as yeast, or the commonly used CHO – Chinese hamster ovary – cells). Sometimes eukaryote-derived proteins will be truncated or otherwise mistranslated by E. coli due to differential codon bias; errors in translation can be prevented by providing additional tRNAs to the culture or directly to the bacteria via plasmids. Despite all this complexity, prokaryotic hosts have been plenty good enough to produce proteins for certain therapies, notably the cytokine G-CSF. This cytokine is taken by patients needing to replenish their white blood cells (e.g., after chemotherapy), and sold as Neupogen by the company Amgen.
-This is it, folks! Moment of truth. Time to find out how the proteins that you worked so hard to express, purify, and test really behave. Although you should be able to produce reasonable titration curves by following the example of Nagai, the introduction/review of binding constants below may help contextualize your analysis.
+===Part 3: Primer design for mutagenesis===
-Let’s start by considering the simple case of a receptor-ligand pair that are exclusive to each other, and in which the receptor is monovalent. The ligand (L) and receptor (R) form a complex (C), which can be written
+[[Image:Sp16 M1D2 primer design schematic.png|thumb|right|300px| '''Primer design schematic for NEB Q5 Site Directed Mutagenesis.''']]
-<center>
+It wouldn’t be very experimentally efficient to somehow pick out and modify a single residue on inverse pericam post-translationally. Instead, researchers genetically encode desired mutations, by making mutated copies of a plasmid that contains the inverse pericam DNA sequence. In addition to non-mutagenic amplification of a specific piece of DNA, synthetic primers can be used to incorporate desired mutations into the DNA. Primer design for site-directed mutagenesis is quite straightforward: the forward primer introduces a mutation into the coding strand. Both non-mutagenic and mutagenic amplification require cycles of DNA melting, annealing, and extension.
-<math> R + L   \rightleftharpoons\ ^{k_f}_{k_r}      C </math>
-</center>
-At equilibrium, the rates of the forward reaction (rate constant = <math>k_f</math>) and reverse reaction (rate constant = <math>k_r</math>) must be equivalent. Solving this equivalence yields an equilibrium dissociation constant <math>K_d</math>, which may be defined either as <math>k_r/k_f</math>, or as <math>[R][L]/[C]</math>, where brackets indicate the molar concentration of a species. Meanwhile, the fraction of receptors that are bound to ligand at equilibrium, often called ''y'' or &theta;, is <math>C/R_{TOT}</math>, where <math>R_{TOT}</math> indicates total (both bound and unbound) receptors. Note that the position of the equilibrium (''i.e.'', ''y'') depends on the starting concentrations of the reactants; however, <math>K_d</math> is always the same value. The total number of receptors <math>R_{TOT}</math>= [''C''] (ligand-bound receptors) + [''R''] (unbound receptors). Thus,
+Remember from Day 1 that primers used in PCR amplification must meet several design criteria in order to ensure specificity and efficiency. Consider the following design guidelines for mutagenesis primers and think about how these differ from the guidelines for non-mutagenic amplification:
-<center>
+*The desired mutation (1-2 bp) must be present in the middle of the forward primer.
-<math>\qquad y = {[C] \over R_{TOT}} \qquad = \qquad {[C] \over [C] + [R]} \qquad = \qquad {[L] \over [L] + [K_d]} \qquad</math>
+*The forward and reverse primers should 'face' away from the mutation and be 'back-to-back' when annealed to the template.
-</center>
+*The primers should be 25-45 bp long.
+*A G/C content of > 40% is desired.
+*Both primers should terminate in at least one G or C base.
+*The melting temperature should exceed 78&deg;C, according to:
+**T<sub>m</sub> = 81.5 + 0.41 (%GC) – 675/N - %mismatch
+**where N is primer length, and the two percentages should be integers.
-where the right-hand equation was derived by algebraic substitution. If the ligand concentration is in excess of the concentration of the receptor, [''L''] may be approximated as a constant, ''L'', for any given equilibrium. Let’s explore the implications of this result:
+To demonstrate primer design, the illustration below uses S101L, which is an uninteresting mutation but is a straightforward teaching example.
-*What happens when ''L'' << <math>K_d</math>?
+<div style="padding: 10px; width: 760px; border: 5px solid #01DFD7;">
-::&rarr;Then ''y'' ~ <math>L/K_d</math>, and the binding fraction increases in a first-order fashion, directly proportional to ''L''.
+Residue 101 of calmodulin is serine, encoded by the AGC codon. This is residue 379 with respect to the entire inverse pericam construct,
+and we can find it and some flanking code in the DNA sequence from Part 2:
-*What happens when ''L'' >> <math>K_d</math>?
+<font face="courier">
-::&rarr;In this case ''y'' ~1, so the binding fraction becomes approximately constant, and the receptors are saturated.
+<small>
-*What happens when ''L'' = <math>K_d</math>?
+(5') GAG GAA ATC CGA GAA GCA TTC CGT GTT TTT GAC AAG GAT GGG AAC GGC TAC ATC AGC GCT (3')
-::&rarr;Then ''y'' = 0.5, and the fraction of receptors that are bound to ligand is 50%. This is why you can read <math>K_d</math> directly off of the plots in Nagai’s paper (compare Figure 3 and Table 1). When ''y'' = 0.5, the concentration of free calcium (our [''L'']) is equal to <math>K_d</math>. '''This is a great rule of thumb to know.'''
-The figures below demonstrate how to read <math>K_d</math> from binding curves. You will find semilog plots (right) particularly useful today, but the linear plot (left) can be a helpful visualization as well. Keep in mind that every ''L'' value is associated with a particular equilbrium value of ''y'', while the curve as a whole gives information on the global equilibrium constant <math>K_d</math>.
+(5') GCT CAG TTA CGT CAC GTC ATG ACA AAC CTC GGG GAG AAG TTA ACA GAT GAA GAA GTT GAT (3')
+</small>
+</font>
-[[Image:20109 Fa15 M2D7 figure.png|thumb|300px|left|'''Simple binding curve.''' The binding fraction ''y'' at first increases linearly as the starting ligand concentration is increased, then asymptotically approaches full saturation (''y''=1). The dissociation constant <math>K_d</math> is equal to the ligand concentration [''L''] for which ''y'' = 1/2.]]
+To change from serine to leucine, one might choose TTA, TTG, or CTN (wherer N = T, A, G, or C). Because CTC requires only two mutations (rather than three as for the other options), we choose this codon.
-[[Image:20109 Fa15 M2D7 figure2.png|thumb|300px|center|'''Semilog binding curves.''' By converting ligand concentrations to logspace, the dissociation constant is readily determined from the inflection point of the sigmoidal curve. The three curves each represent different ligand species. The middle curve has a <math>K_d</math> close to 10 nM, while the right-hand curve has a higher <math>K_d</math> and therefore lower affinity between ligand and receptor (vice-versa for the left-hand curve).]]
-<br style="clear:both;"/>
-Of course, inverse pericam has multiple binding sites, and thus IPC-calcium binding is actually more complicated than the example above. The <math>K_d</math> reported by Nagai is called an ‘apparent <math>K_d</math>’ because it reflects the overall avidity of multiple calcium binding sites, not their individual affinities for calcium. Normally, calmodulin has a low affinity (N-terminus) and a high affinity (C-terminus) pair of calcium binding sites. However, the E104Q mutant, which is the version of CaM used in inverse pericam, displays low affinity binding at both termini. Moreover, the Hill coefficient, which quantifies cooperativity of binding in the case of multiple sites, is reported to be 1.0 for inverse pericam. This indicates that inverse pericam behaves as if it were binding only a single calcium ion per molecule. Thus, wild-type IPC is well-described by a single apparent <math>K_d</math>.
+Now we must keep >10 bp of sequence on each side in a way that meets all our requirements. To quickly find G/C content and see secondary structures, look at the [http://www.idtdna.com/calc/analyzer IDT website]. (Note that the T<sub>m</sub> listed at this site is '''''not''''' one that is relevant for mutagenesis.)
-For any given mutant, things may be more complicated. Keep in mind that we are not directly measuring calcium binding, but instead are indirectly inferring it based on fluorescence (for both mutant and wild-type IPC). A change in fluorescence requires the participation not only of calcium, but also of M13. In addition to the four separate calcium binding sites in calmodulin, the M13 binding site influences apparent affinity and apparent cooperativity. In short, be careful about how you describe the meanings of our binding parameters in your reports.
+Ultimately,  your forward primer might look like the following, which has a T<sub>m</sub> of almost 81&deg;C, and a G/C content of ~58%.
-Returning to the big picture: when you write your Protein engineering summary, be sure to consider how changes in both binding affinity and cooperativity (and even potentially raw fluorescence differences) can affect the practical utility of a sensor.
+<font face="courier">
+’ GG AAC GGC TAC ATC CTC GCT GCT CAG TTA CGT CAC G 3'
+</font><br>
-==Protocols==
+The reverse primer is the inverse complement of a sequence just preceding the forward primer in the IPC gene.  The forward and reverse primers are set up back-to-back.
+</div style>
-===Part 2: Prepare samples for titration curve===
+Lucky for us, NEB has a tool that can design our mutagenic primers.
+#Go to the [http://nebasechanger.neb.com/ NEBaseChanger] site and click 'Please enter a new sequence to begin.'
+#*A new window will open.  Copy and paste the wild-type IPC sequence.
+#Confirm that the 'Substitution' option is selected.
+#Highlight the basepairs you want to mutate using by scrolling through the sequence, or you can search the sequence by typing the basepairs into the 'Find' box.
+#Type the new DNA sequence (the basepair(s) you want your forward mutagenic primer to incorporate into the IPC sequence) in the 'Desired Sequence' box.
+#*Under the Result header, a diagram showing where your primers will anneal is provided.
+#*Under the Required Primers header, the sequences for your forward primer and reverse primer are shown with the characteristics for each.
+#Screen capture the information provided in the Result and Required Primers sections.
+#*Embed the images in your notebook.
+#*Print the screen capture and submit it to the teaching faculty before you leave today.  In addition, record your primer sequences in the table on the [http://engineerbiology.org/wiki/Talk:20.109%28S16%29:Design_mutation_primers_%28Day2%29 Discussion] page.
+#*It is '''very important''' that you submit your primer sequences before you leave!  The teaching faculty will order your primers from IDT DNA tonight to ensure they arrive by your next class.
+#Use the guidelines above to examine the mutagenesis primers designed by NEBaseChanger.  Include your thoughts in your notebook.
+#*Do NOT alter the primers provided by NEB.
-==== Tips for success====
+===Part 2: Primer preparation===
+While you were away the sequences for the mutagenic primers you designed were submitted to Integrated DNA Technologies (IDT).  IDT synthesized the primers then lyophilized (dried) them to a powder.  Follow the steps below to resuspend your primers.
+#Centrifuge the tubes containing your lyophilized primers for 1 min.
+#Calculate the amount of water needed ''for each primer'' (forward and reverse, separately) to give a concentration of 100 &mu;M.
+#Resuspend each primer stock in the appropriate volume of sterile water, vortex, and centrifuge.
+#Now prepare a dilution from your archival stock. Prepare 100 &mu;L of a solution that has both the forward and reverse primers,  ''each'' primer at 10 &mu;M.
+#*Try the calculation on your own first.  If you get stuck, ask the teaching faculty for help.
+#*Be sure to change tips between primers!
+#Return the rest of your primer stocks, plus your primer specification sheets, to the front bench.
-Take great care today to limit the introduction of bubbles in your samples. When expelling fluid, pipet '''''slowly''''' while touching the pipet tip against the bottom or side of the well.
+===Part 3: Site-directed mutagenesis===
-<!--
-When using the multichannel pipet, always check to make sure all tips are getting filled - sometimes one tip may not be on all the way, and will pull up less volume than the others. If this happens, release the fluid, adjust the tip, and try again.
--->
-====Protocol====
+[[Image:Sp16 M1D3 SDM schematic.png|thumb|center|550px|'''Schematic of NEB Q5 Site Directed Mutagenesis Kit procedure modified from NEB manual.''']]
-[[File:Fa15 Protein Ca assay plate map.png|thumb|450px|right|Titration plate map]]
+We will be using the Q5 Site Directed Mutagenesis Kit from NEB to perform your site-directed mutagenesis reactions. Each group will set up one reaction, for your X#Z mutation. Meanwhile, the teaching faculty will set up a single positive control reaction, to ensure that all the reagents are working properly. You should work quickly but carefully, and keep your tube in a chilled container at all times. '''Please return shared reagents to the ice bucket(s) from which you took them as soon as you are done with each one.'''
-#Take a black 96-well plate, and familiarize yourself with the plate map scheme at right: top two rows are to be loaded with wild-type IPC, next two rows are to be loaded with your X#Z mutant IPC, and the final row is to be loaded with  water/BSA to serve as a blank/background row.
+#Get a PCR tube and label the top with your mutation and lab section (write small!).
-#*The dark sides of the plate reduce "cross-talk" (''i.e.'', light leakage) between samples in adjacent wells, another potential contribution to error.
+#Add 10.25 &mu;L of nuclease-free water.
-#Aliquot your wild-type protein to your plate. Use your P200 pipet to add 30 μL of protein (per well) to rows A and B of your plate.
+#Add 1.25 μL of your mutagenesis primer mix (each primer should be at a concentration of 10 &mu;M).
-#Aliquot you X#Z mutant IPC to your plate.  Use your P200 pipet and add 30 μL of protein (per well) to rows C and D of your plate.
+#Add 1 &mu;L of IPC template DNA (concentration of 25 ng/&mu;L).
-#Finally, add 30 μL of water with only 0.1% BSA (no IPC) to row 5(E) of your plate.
+#Lastly, use a filter tip to add 12.5 &mu;L of Q5 Hot Start High-Fidelity 2X Master Mix - containing buffer, dNTPs, and polymerase - to your tube.
-#The calcium solutions are at the front bench in shared reservoirs.  '''Carefully''' carry your plate to the front bench to add these solutions with the  multi-channel pipet.
+#Once all groups are ready, we will begin the thermocycler, under the following conditions:
-#Using shared reservoir #1 (lowest calcium concentration - actually 0 nM), add 30 μL to the top five rows in the first column of the plate. Discard the pipet tips.
-#Now work your way from reservoirs #2 to #12 (highest calcium concentration), and from the left-hand to the right-hand columns on your plate. Be sure to use fresh pipet tips each time! If you do contaminate a solution, let the teaching faculty know so they can put out some fresh solution. Honesty about a mistake is far preferred here to affecting every downstream experiment.
-#When you are done, alert the teaching faculty and you will be taken in small groups to measure the fluorescence values for your samples.
-===Part 3: Fluorescence assay===
+<center>
+{| border="1"
+! Segment
+! Cycles
+! Temperature
+! Time
+|-
+| Initial denaturation
+| 1
+| 98 &deg;C
+| 30 s
+|-
+| Amplification
+| 25
+| 98 &deg;C
+| 10 s
+|-
+|
+|
+| 55 &deg;C
+| 30 s
+|-
+|
+|
+| 72 &deg;C
+| 2 min
+|-
+| Final extension
+| 1
+| 72 &deg;C
+| 2 min
+|-
+| Hold
+| 1
+| 4 &deg;C
+| indefinite
+|}
+</center>
-#The BMC (BioMicro Center) has graciously agreed to let us use their plate reader. Walk over to building 68 with a member of the teaching staff.
+*After the cycling is completed, the teaching faculty will complete the KLD reaction (which stands for "kinase, ligase, ''DnpI''") using 1 &mu;L of your amplification product, 5 &mu;L 2X KLD Reaction Buffer, 1 &mu;L KLD Enzyme Mix, and 3 &mu;L nuclease-free water. The reactions will be incubated for 5 min at room temperature.
-#You will be shown how to set the excitation (485 nm) and emission (515 nm) wavelength on the plate reader to assay your protein.
-#Your raw data will be posted on today's [[Talk:20.109(S16):Characterize protein expression (Day7)|Discussion page]] and emailed to you as a .txt file so you can begin your analysis.
-You will analyze your calcium titration assay data in two steps. First, you will get a rough feel for how your mutant changed (or didn't) compared to wild-type IPC by plotting the two replicate values and their average, in both raw and processed form. Second, you will take the average processed values and plug them into some <small>MATLAB</small> code that will more precisely tell you the affinity and cooperativity of each protein with respect to calcium.
+*The teaching faculty will then use 5 &mu;L of the KLD reaction product to complete a transformation into an ''E. coli'' strain (NEB 5&alpha; cells of genotype ''fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17'') that will amplify the plasmid such that you are able to confirm the appropriate mutation was incorporated.  The transformation procedure will be as follows:
+#Add 5 &mu;L of KLD mix to 50 &mu;L of chemically-competent NEB 5&alpha;.
-===Part 1: Titration curve in Excel and first estimate of ''K<sub>d</sub>''===
+#Incubate on ice for 30 min.
+#Heat shock at 42 &deg;C for 30 s.
-Today  you will analyze the fluorescence data that you got last time. Begin by analyzing the wild-type protein as a check on your work (your curve should resemble Nagai's Figure 3L), and then move on to your mutant samples. If you are not familiar with manipulations in Excel, use the ''Help'' menu or ask the teaching faculty for assistance.
+#Incubate on ice for 5 min.
+#Add 950 &mu;L SOC and gently shake at 37 &deg;C for 1 h.
-#Open an Excel file for your data analysis. Begin by making a column of the free calcium concentrations present in your twelve test solutions. Assuming a 1:1 dilution of protein with calcium, the final concentrations are: 0 nM, 8.5 nM, 19 nM, 32.5 nM, 50 nM, 75 nM, 112.5 nM, 175 nM, 301 nM, 675 nM, 1.505 &mu;M, 19.5 &mu;M. Be sure to convert all concentrations to the same units.<br>
+#Spread 50 &mu;L onto LB+Amp plate and incubate overnight at 37 &deg;C.
-#Now open the text file containing your raw data as a tab-delimited file in Excel (you can download the file from the  [[Talk:20.109(S16):Characterize protein expression (Day7) | M1D7 Discussion]] page). Convert the row-wise data to column-wise data (using ''Paste Special'' &rarr; ''Transpose''), and transfer each column to your analysis file. Add column headers to indicate which protein is which, and analyze each replicate separately for now. Also include a column of your control samples that did not contain protein.
-#Begin by calculating the average of your blank samples, and bold this number for easy reference. It is the background fluorescence present in the calcium solutions and should be quite low. If necessary, subtract this background value from each of your raw data values. It may help to have a 6-column series called “RAW”, and another called “SUBTRACTED.”
-#Next you should normalize your data. The maximum and minimum fluorescence values for a given titration series should be defined as 100% and 0% fluorescence, respectively, and every other fluorescence value should be expressed as a percentage in between. Think about how to mathematically express these conditions.
-#*First calculate the percent fluorescence for both replicates. Then make a new column and calculate the average percentage as well.
-#*Alternatively, average your data first, and then normalize the average data.  How do the "average then normalize" and the "normalize then average" curves compare?  Which one will you include in your Protein engineering summary?
-#*If one data point seems really off from the other replicate and from the expected trend, you might consider it an outlier and delete it, especially if you have good reason to believe that there was a reason (error in pipetting, air bubble in that well) for the anomaly. Otherwise, you might be losing valuable information, and/or misleading anyone who tries to interpret your data.
-#For each protein, plot this normalized data versus calcium concentration. Save these plots in case you want to include them in your report.
-#*You might plot the two replicates as points and their average value as a dashed line (see [http://engineerbiology.org/wiki/20.109%28S16%29:Module_1 front page] of this module).
-#Note down the approximate inflection points of the curves, which should occur at half-saturation: these indicate the approximate values of the apparent <math>K_d</math> for each sample.
-===Part 2: Improved estimate of ''K<sub>d</sub>'' using <small>MATLAB</small> modeling===
-====Preparation====
-#Download these three files: [[Media:F15 Fit Main.m  | F15_Fit_Main]], [[Media:Fit_SingleKD.m| Fit_SingleKD]], and [[Media:Fit_KDn.m| Fit_KDn]]. Move them to the username/Documents/MATLAB folder on your computer.
-# Double-click on the <small>MATLAB</small> icon to start up this software.
-# The main window that opens is called the command window: here is where you run programs (or directly input commands) and view outputs. You can also see and access the command history, workspace, and current directory windows, but you likely won’t need to today.
-# In the command window, type ''more on''; this command allows you to scroll through multi-page output (using the spacebar), such as help files.
-# In addition to the command area, <small>MATLAB</small> comes with an editor. Click ''File'' &rarr; ''Open'' and select the program '''F15_Fit_Main'''. It has the .m extension and thus is executable by <small>MATLAB</small>. Read the introductory comments (the beginning of a comment is indicated by a % sign), and then input your fluorescence data.
-# Read through the program, and as you encounter unfamiliar terms, return to the workspace and type ''help functionname''. Feel free to ask questions of the teaching faculty as well.
-#* You might read about such built-in functions as ''logspace'' and ''nlinfit''.
-#* You will also want to open and read '''Fit_SingleKD''' – a user-defined function called by '''F15_Fit_Main''' – in the <small>MATLAB</small> editor.
-#* If you type ''help function'' you will learn the syntax for a function header.
-#* Note that a dot preceeding an operator (such as A ./ B or A .* B) is a way of telling <small>MATLAB</small> to perform element-by-element rather than matrix algebra.
-#* Also note that when a line of code is ''not'' followed by a semi-colon, the value(s) resulting from the operation will be displayed in the command window.
-====Analysis====
-# Once you more-or-less follow Part 1 of the program, type '''F15_Fit_Main''' in the workspace, hit return to run the program, and consider the following questions:
-#* Why must the fluorescence data be transformed (from ''S'' to ''Y'') prior to using in the model?
-#* What <math>K_d</math> values are output in the command window, and how do they compare to the values you estimated from your Excel plots?
-#* Figure 1 should display your wild type and mutant data points and model curves. How do they look in comparison to the curves you plotted in Excel?
-#* Figure 2 should display the residuals (difference between data and model) for your three proteins. If the absolute values are low, this indicates good agreement between the model and the data numerically. Whether or not this is the case, another thing to look for is whether the residuals are evenly and randomly distributed about the zero-line. If there is a pattern to the errors, likely there is a systematic difference between the data and the model, and thus the model does not reflect the actual binding process well. What are the residuals like for each of your modeled proteins?
-# Now move on to Part 2 of the '''F15_Fit_Main''' program. Part 2 also fits the data to a model with a single, ‘apparent’ value of <math>K_d</math>, but it allows for multiple binding sites and tests for cooperativity among them. The parameter used to measure cooperativity is called the Hill coefficient. A Hill coefficient of 1 indicates independent binding sites, while greater or lesser values reflect positive or negative cooperativity, respectively. Let the following questions guide you as you proceed:
-#*Visually, which model appears to fit your wild-type data better (Fig. 3 ''vs.'' Fig. 1)? Your mutant data?
-#*Do the respective residuals support your qualitative assessment (Fig. 4 ''vs.'' Fig. 2)?
-#*Numerically, how do the values of <math>K_d</math> compare for the two models? How does the value of ''n'' compare to the implicitly assumed value of 1 in Part 1?
-#*Do you see changes in binding affinity and/or cooperativity between the wild-type and X#Z samples? Do they match your ''a priori'' predictions?
-#*'''Don't forget to save any figures you want to use in your report!''' If the legends are covering up your data, you can simply move them over with your mouse.
-# Finally, you can skim Part 3 of the '''F15_Fit_Main''' program. Make sure you update the range of the linear transition region for each IPC sample, but beyond this, don’t worry too much about the coding details; rather do read through the comments.
-#* Look at Part 1 of Figure 5: are the binding curves asymptotic, sigmoidal, or other? What does this shape indicate? You can use the zoom button to get a closer look at part of the plot, or the ''axis'' command present in the code. (Don't worry too much about this question if it is unclear.)
-#* Now look in the command window. What values of <math>K_d</math> and Hill coefficient (''n'') do you get for your three proteins? How do the <math>K_d</math>’s from Part 3 compare to the ones from Parts 1 and 2? Don’t be discouraged if your wild-type values do not exactly match Nagai’s work, or if there is variation between Parts 1, 2, and 3.
-#*Comparing the model and data points by eye (Part 2 of Figure 5), do you think it is a good model for any of your proteins? If so, which ones? What experimental limitations might prevent Hill analysis from working well, especially for some mutants?
-#*Why should only the transition region be analyzed in a Hill plot?
-#* What is the relationship between slope and <math>K_d</math> and/or ''n'', and intercept and <math>K_d</math> and/or ''n''?
-#If your mutant proteins are not well-described by any of the models so far, what kind of model(s) (qualitatively speaking) do you think might be useful?
-#*Optional: If your data might be well-described by a model with two <math>K_d</math>'s (or if you are interesting in exploring some sample data that is), download and run [[Media:Fit_TwoKD.m | Fit_TwoKD]] and [[Media:Fit_TwoKD_Func.m | Fit_TwoKD_Func]].
 ==Reagent list==
+*Q5 Site Directed Mutagenesis Kit from NEB
+**Q5 Hot Start High-Fidelity 2X Master Mix
-*Calcium calibration kit from Life Technologies
+***Propriety mix of Q5 Hot Start High-Fidelity DNA Polymerase, buffer, dNTPs, and Mg<sup>2+</sup>.
-**Zero free calcium buffer: 10 mM EGTA in 100 mM KCl, 30 mM MOPS, pH 7.2
+**2X KLD Reaction Buffer
-**39 &mu;M free calcium buffer: 10 mM CaEGTA in 100 mM KCl, 30 mM MOPS, pH 7.2
+**10X KLD Enzyme Mix
+***Proprietary mix of kinase, ligase, and ''DpnI'' enzymes.
-*Thermo Scientific Varioskan Flash Spectral Scanning Multimode Reader
+*The SOC medium contains 2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose.
+*LB+Amp plates
+**The Luria-Bertani (LB) broth contains 1% tryptone, 0.5% yeast extract, and 1% NaCl
+**Plates prepared by adding 1.5% agar and 100 μg/mL ampicillin to LB
 ==Navigation links==
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