Now that my lab is fully equipped, I’m taking on rotation students. Unfortunately, with the pandemic, it’s harder to have one-on-one meetings where I can sit down and walk the new students through every method. Furthermore, why repeat teaching the same thing to multiple students when I can just make an initial written record that everyone can reference and just ask me questions about? Thus, here’s my instructional tutorial on how I design primers in the lab. 12/2/24 update: Don’t forget to look at the most up-to-date strategy described at the bottom of the page!

First, it’s good to start out by making a new benchling file for whatever you’re trying to engineer. If you’re just making a missense mutation, then you can start out by copying the map for the plasmid you’re going to use as a template. Today, we’ll be mutating a plasmid called “G619C_AttB_hTrim-hCPSF6(301-358)-IRES-mCherry-P2A-PuroR” to encode the F321N mutation the CPSF6 region. This should abrogate the binding of this peptide to the HIV capsid protein. Eventually every plasmid in the lab gets a unique identifier based on the order it gets created (this is the GXXXX name). Since we haven’t actually started making this plasmid yet, I usually just stick an “X” in front of the name of the new file, to signify that it’s *planned* to be a new plasmid, with G619C being used as the template. Furthermore, I write in the mutation that I’m planning to make in it. Thus, this new plasmid map is now temporarily being called “XG619C_AttB_hTrim-hCPSF6(301-358)-F321N-IRES-mCherry-P2A-PuroR”

That’s what the overall plasmid looks like. We’ll be mutating a few nucleotides in the 4,000 nt area of the plasmid.

I’ve now zoomed into the part of the plasmid we actually want to mutate. The residue is Phe321 in the full length CPSF6 protein, but in the case of this Trim-fusion, it’s actually residue 344.

I next like to “write in” the mutation I want to make, as this 1) makes everything easier, and 2) is part of the goal of making a new map that now incorporates that mutation. Thus. I’ve now replaced the first two T’s of the Phe codon “TTT” with two A’s, making the “AAT” codon which encodes Asn (see the image above)

Next is planning the primers. So there are a few ways one could design primers to make the mutation. I like to create a pair of overlapping (~ 17 nt), inverse primers, where one of the primers encodes the new mutation in it. PCR amplification with these primers should result in a single “around-the-circle” amplicon, where there is ~ 17 nt of homology on the terminal ends. These ends can then be brought together and closed using Gibson assembly.

So first to design the forward primer. This is the primer that will go [5’end] –[17 nt homology] — [mutated codon] — [primer binding region] — [3’end]. So the first step is to figure out the primer binding region.

In a cloning scheme like this, I like to start selecting the nucleotides directly 3′ of the codon to be mutated, and select enough nucleotides such that the melting temperature is ~ 55*C. In actuality, the melting temperature will be slightly higher, since 1) we will end up having 17 nt of matching sequence 5′ of the mutated codon, and 2) the 3rd nt in the codon, T, will actually be matching as well.

Now that I’ve determined how long I need that 3′ binding region to be, I select the entire set of nucleotides I want in my full primer. In this case, this ended up being a primer 36 nt in length (see below).

Since this is the forward primer, I can just copy the “sense” version of this sequence of nucleotides.

OK, so next to design the reverse primer. This is simpler, since it’s literally just a series of nucleotides going in the antisense orientation directly 5′ of the codon (as it’s shown in the sense-stranded, plasmid map). I shoot for ~ 55*C to 60*C, usually just doing a little bit under 60*C.

Since this is the reverse primer, we want the REVERSE COMPLEMENT of what we see on the plasmid map.

Voila, we now have the two primers we need. We just now need to order these oligos (we order from ThermoFisher, since it’s the cheapest option at CWRU, and can then perform the standard MatreyekLab cloning workflow).

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12/2/24 update: So the above instructions work fine, but I have since (as in like 3/4 years ago, haha) adopted a slightly different strategy. The original strategy prioritized having one primer (in the above example, the reverse primer, or KAM3402) being non-mutagenic / perfectly matching the WT sequence, so that it could double as a Sanger sequencing primer in other non-cloning circumstances. Well, we barely ever need these anymore, especially with the existence of whole-plasmid nanopore sequencing via Plasmidsaurus. Thus, I now:
1) Just design the primer pairs so that both the forward AND reverse primers are each encoding the mutated nucleotides, since that likely better balances primer Tm’s. To get the ideal 18+ nt of homology necessary for Gibson, I then append ~ 9 nt of matching sequence to the 5′ ends of each primer (so 9 matching nucleotides to the “left” of the mutated portion).
2) I also now like to reduce the anticipated Tm of the 3′ binding portion of the primer (so everything to the “right” of the mutated portion) to ~ 50-55*C, since there’s going to be some amount of binding energy provided by the ~ 9 nt of matching sequence on the other side of the primer.

Thus, my updated primer strategy for the above reactions would look like this:

I do a lot of molecular cloning, which means a lot of transformations of chemically competent e.coli. Using 50 uL of purchased competent bacteria would cost about $10 per transformation, which would be an AWFUL waste of money, especially with this being a highly recurring expense in the lab. I had never made my own competent cells before, so I had to figure this out shortly after starting my lab. It took a couple of days of dedicated effort, but it ended up being quite simple (I’ll link to my protocol a bit later on). Though my frozen stocks ended up working fine, I became quite used to creating fresh cells every time I need to do a transformation. The critical step here is taking a saturated overnight starter culture, and diluting it so you can harvest a larger volume of log-phase bacteria some short time later. A range of ODs [optical density here defined as absorbance at 600 nm] work, though I like to use bacteria at an OD around 0.2. I had gotten pretty good at being able to eyeball when a culture was ready for harvesting (for LB in a 250 mL flask, I found this was right when I started seeing turbidity), but I figured there was a better way to know when it’s worth sampling and harvesting.

I started keeping good notes about 1) the starting density of my prep culture (OD of the overnight culture divided by the dilution factor), 2) the amount of time I left the prep culture growing, and 3) the final OD the prep culture. I converted everything into cell density which is a bit more intuitive than OD (I found 1 OD[A600] of my bacteria roughly corresponded to 5e8 bacteria per mL), and worked in those units from there on out. Knowing bacteria exhibit exponential growth, I log base-10 transformed the counts. Much like the increasing number of COVID-19 deaths experienced by the US from early March through early April, exponential growth becomes linear in log-transformed space. I figured I could thus estimate the growth of my prep culture of competent cells by making a multi-variate linear model, where the final density of the bacteria was dependent on the starting bacterial density and how long I left it growing. I figured the lag-phase from taking the saturated culture and sticking it into cold-LB would end up being a constant in the model. Here’s my dataset, and here’s my R Markdown analysis script. My linear model seemed to perform pretty well, as you can see in the below plot. As of writing this, the Pearson’s r was 0.98.

The aforementioned analysis script has a final chunk that allows you to input the starting OD of your starter culture, and assuming a 1000-fold dilution, tells you how long you likely need to wait to hit the right OD of your prep culture. Then again. I don’t think anyone really wants to enter this info into a computer every time they want to set up a culture, so I made a handy little “look-up plot”, shown below, where a lab member could just look at their starter culture OD on the x-axis, choose the dilution they want to do (staying 2x within 1000-fold since I don’t know if smaller dilutions can affect bacterial competency), and figure out when they need to be back to harvest (or at least stick the culture on ice). I’ve now printed this plot out and left it by my bacterial shaker-incubator.

Note: The above data was collected when diluting starter culture bacteria into *COLD* LB that was stored in the fridge. We’ve since shifted to diluting the bacteria into room-temp LB (~ 25*C), which has somewhat expectedly resulted in slightly faster times to reach the desired OD. If you’re doing that too, I would suggest subtracting ~ 30min of incubation time from the above times to make sure you don’t overshoot your desired OD.

I’m still much more of a wet-lab scientist than a computational one. That said, god damn do I still think the moderate amount of computational work I can do is still empowering.

I only learned about Gibson when I started my postdoc, and it completely changed how I approached science. In some experiments with Ethan when I was in the lab, I was blown away when we realized that you don’t even need Gibson mix to piece a plasmid back together; this is something we were exploring to try to figure out if we could come up with an easier & more economical library generation workflow. I was disappointed but equally blown away when I realized numerous people had repeatedly “discovered” this fact in the literature already; the most memorable of the names given to it was IVA, or In-Vitro Assembly. Ethan had tried some experiments, and had said it worked roughly as well as with Gibson. Of course, I can’t recall exactly what his experiment was at this point (Although probably a 1-piece, DNA recircularization reaction, since this was in the context of inverse PCR-based library building, after all). So the take away I had was that it was a possible avenue for molecular cloning in the future.

We’ve done a fair amount of molecular cloning in the lab already, creating ~ 60 constructs in the first 4 months since Sarah joined. I forgot exactly the circumstances, but something was right where it made sense to try some cloning where we didn’t add in Gibson mix. I was still able to get a number of intended constructs on that first try, so I stuck to not adding Gibson mix for a few more panels of constructs. I’ve been trying to keep very organized with my molecular cloning pipelines and inventories, which included keeping track of how often each set of mol cloning reactions yielded correctly pieced-together constructs. I’ve taken this data, and broken it down based on two variables: whether it was a 1- or 2-part DNA combination (I hardly ever try more than 2 in a single reaction, for simplicities’ sake, and also because properly combined cloning intermediates may still be useful down the line, anyway), and whether Gibson mix was added or not. Here’s the current results:

So this was extremely informative. Some points
1) I’m willing to screen at least 4 colonies for a construct I really want. Thus, I’m counting a success rate > 0.25 as being a “successful” attempt at cloning a construct. In the above plot, that means any area above the dotted red line. Thus, 1-part DNA recircularizations have pretty decent success rates, since the area of the colored curve above the red dotted like >> the area below it. Sure, Gibson mix helps, but it’s not a night-and-day difference.
2) 2-part DNA combinations are a completely different story,. Lack of Gibson means that I have just as many failed attempts at cloning something as successful attempts. Those are not great odds. Adding Gibson mix makes a big difference here, since it definitely pushes things in favor of a good outcome. Thus, I will ALWAYS be adding GIbson mix before attempting any 2-part DNA combinations.

Other notes: I’m using home-grown NEB 10-beta cells, which give me pretty decent transformation rates (high-efficiency 1-part recircularization reactions can definitely yield many hundreds of colonies on the plate from a successful attempt), so there have been relatively few plates where I literally have ZERO colonies, where I’m more likely to have a few colonies that are just hard-to-remove residual template DNA).

So as I bring in trainees into the lab, I’ll want them to learn how to do some (at the very least) basic data analyses. I realize they may not know exactly where to start, so as I go about making my own basic analysis scripts for analyzing data relevant to me, I’ll post them here and make sure they’re reasonably well commented to explain what is going on.

OK, the specific backstory here. Back in UW, we had next-day IDT orders. It became very clear that was not going to be the case at CWRU, especially after talking to the IDT rep (who did not seem to really care about having a more thriving business here). So, I priced out my options, and Thermo ended up handedly winning the oligo price battle ($0.12 a nucleotide. which is a slight improvement over the $0.15 we seemed to be paying at UW *Shrug*). Thermo also does no shipping cost (another bonus), with the downside being that they only deliver on Tuesdays and Thursdays. I wanted to figure out how long it takes to receive primers are ordering them, so I’ve been keeping track of when I made each order, and how long it took to arrive. Now that I have a decent number of data-points, I decided to start analyzing it to figure out if there were any patterns emerging.

Here’s a link to the data. Here’s a link to the R Markdown file. You’ll want both the data and the R Markdown file in the same directory. I’m also copy-pasting the code below, for ease:

Primer_wait_analysis

This is the first chunk, where I’m setting up my workspace by 1) Starting the workspace fresh 2) Importing the packages I’ll need 3) Importing the data I’ll need

# I always like to start by clearing the memory of existing variables / dataframes / etc.

rm(list = ls())

#Next, let's import the packages we'll need.
#1) Readxl, so that I can import the excel spreadsheet with my data
#2) Tidyverse, for doing the data wrangling (dyplr) and then for plotting the data (ggplot)

library(readxl)
library(tidyverse)

## ── Attaching packages ─────────────────────────────────────────────────────────────────────────── tidyverse 1.3.0 ──

## ✓ ggplot2 3.2.1     ✓ purrr   0.3.3
## ✓ tibble  2.1.3     ✓ dplyr   0.8.3
## ✓ tidyr   1.0.0     ✓ stringr 1.4.0
## ✓ readr   1.3.1     ✓ forcats 0.4.0

## ── Conflicts ────────────────────────────────────────────────────────────────────────────── tidyverse_conflicts() ──
## x dplyr::filter() masks stats::filter()
## x dplyr::lag()    masks stats::lag()

# Lastly, use readxl to import the data I want

primer_data <- read_excel("How_long_it_takes_to_receive_items_after_ordering.xlsx")

The idea is to make a bar-graph (or equivalent) showing how many days it takes for the primers to arrive depending on what day I order the primers. I first have to take the raw data and do some wrangling to get it in the format I need for plotting.

# I care about how long it took me to receive primers in a *normal* week, and not about what I encountered during the holidays. Thus, I'm going to filter out the holiday datapoints.

primer_data_normal <- primer_data %>% filter(holiday == "no")

# Next, I want to group data-points based on the day of the week

primer_data_grouped <- primer_data_normal %>% group_by(day_of_week) %>% summarize(average_duration = mean(days_after_ordering), standard_deviation = sd(days_after_ordering), n = n())

# Now let's set the "day_of_week" factor to actually follow the days of the week

primer_data_grouped$day_of_week <- factor(primer_data_grouped$day_of_week, levels = c("M","T","W","R","F","Sa","Su"))

# Since I'll want standard error rather than standard deviation, let's get the standard error
primer_data_grouped$standard_error <- primer_data_grouped$standard_deviation / sqrt(primer_data_grouped$n)

Now that the data is ready, time to plot it in ggplot2.

Primer_plot <- ggplot() + geom_point(data = primer_data_grouped, aes(x = day_of_week, y = average_duration)) +
  geom_errorbar(data = primer_data_grouped, 
                aes(x = day_of_week, ymin = average_duration - standard_error, 
                    ymax = average_duration + standard_error), width = 0.5) + 
  geom_text(data = primer_data_grouped, aes(x = day_of_week, y = 0.2, label = paste("n=",primer_data_grouped$n))) + geom_hline(yintercept = 0) + scale_y_continuous(limits = c(0,7), expand = c(0,0.01)) +
  theme_bw() + xlab("Day primer was ordered") + ylab("Days until primer arrived") + theme(panel.grid.major.x = element_blank())

ggsave(file = "Primer_plot.pdf", Primer_plot, height = 4, width = 6)
Primer_plot

Hmm, probably should have made that figure less tall. Oh well.

So the n values are still rather small, but it looks like I’ll get my primers soonest if I order on Monday or maybe Tuesday. In contrast, ordering on Thursday, Friday, or Saturday give me the longest wait (though well, some of that are the weekend days, which don’t matter as much). Thus, if I have any projects coming up where I have to design primer, it’s probably worth me taking the time to do that on a Sunday or Monday night instead of late in the workweek.

UPDATE 2/22/2020: Uhhh, so the plot above was my first draft attempt, and I have since honed in on the right representation of hte data:

And that’s because average numbers are only somewhat meaningful, and the distribution of frequencies is much more relevant / accurate. Here’s a link to the updated script for generating the plot with the data file at this other link.

Installing Enrich2 / using a Conda environment

I have a couple of new Macs in the lab that need Enrich2 installed. The goal today is to go through the steps of making a Conda environment specifically for running Enrich2 (and installing Enrich2), developed by Alan Rubin.

The Enrich2 repository

The Enrich2 docs

Alan instructions for making a Conda environment specifically for Enrih2

Alan’s instrucitons for doing this is pretty good, if I remember correctly. But, Alan is a seasoned programmer / computational scientist, while people like me are novices and far less familiar with these steps. Furthermore, depending on the specifics of your computer / system, you may get different errors in the installation process. Thus, here’s my interpretation of this process for the benefit of others like me.

0) This supposes that you have already installed an updated version of Anaconda on your Mac. Go back and do this now, if you haven’t done so already.

1) Download the Anaconda Enrich2 environment file and put it somewhere you can access using Terminal.

2A) Update to the newest version of Anaconda, just in case. Hit “y” if prompted.

conda update conda

2B) OK, so now was installing the right version of Pandas it needs. To do this, I first went into Anaconda Navigator and made a new environent called python2, that uses Python 2. Then in terminal, I called:

conda activate python2

2C) This activated python2, so that now the prompt didn’t say “base”, but now it said “(python2)”. Cool. Envinroment activated. I then installed pandas 0.19.2 by typing in:

pip install pandas==0.19.2

2D) OK, this seemed to work too. I got an error when trying to re-run the “enrich2_env.yml file”, so I removed the “=0.19” part form the .yml file and ran “conda env create -f enrich2_env.yml”. This actually seemed to work giving me a list of packages being extracted. Now to actually test it out.

conda activate enrich2

3) Cool, that worked, and the terminal prompt now says “(enrich2)”. Now we’re in business. Next is actually installing Enrich2 now that we’re in the enviroment. Go to the Enrich2 repository, download the file, unzip it, and then move to its directory in terminal. Then run:

python setup.py install

That seemed to work since it didn’t throw any errors.

4) Next is actually trying to run the application. Type in the following:

enrich_gui

Oh Jesus christ. It literally crashes the Finder due to the following errors:

CGSTrackingRegionSetIsEnabled returned CG error 268435459
CGSTrackingRegionSetIsEnabled returned CG error 268435459
CGSTrackingRegionSetIsEnabled returned CG error 268435459
HIToolbox: received notification of WindowServer event port death.
port matched the WindowServer port created in BindCGSToRunLoop

Looks like this may have been a problem with the MacOS operating system. Updating my OS to Catalina and then trying again.

5) OK, MacOS has been updated to Catalina. Now let’s try running Enrich again. (You’ll likely want to run this command after you’ve navigated to the directory with your raw files to simplify the file locating process).

enrich_gui

Awesome. It worked!

PS. I followed these instructions for my second Mac and it worked like a charm. I even went straight for the Cataline update early on and didn’t run into the error in Step4.