Nothing Worked, but That's OK

Having experiments fail is really disheartening.  You spend all this time, sometimes months, trying to set up an experiments.  You order all the supplies and get everything perfectly into place, and when you finally go to test this beautiful idea, it fails. 

It’s very easy to feel disheartened and like you’ve just wasted all those months of planning and preparation only to go back to the drawing board. 

Mod2 was a lesson in taking failure and learning from it.  Our CRISPRi system didn’t just not work, it REALLY didn’t work.  The condition where we expected the most lactate production showed up with a really large negative production (which we made 0, so it didn’t end up looking too bad in the end).  I started to feel bad for failing the experiment, but then I heard from other groups that their CRISPRi systems also didn’t work.

Even though a lot of the experiments didn’t work the way we planned, they provided a good learning experience.  The failure of our experiments forced us to determine why our experiments failed.  We had to think more and synthesize the information we had.  Therefore, we had a better understanding of the topic than when we started.  Failure, even though it can be frustrating, provides an opportunity to deepen our knowledge of a subject so that we can perform even better the next time.

when nothing worked

I came into Mod 2 really excited because CRISPR was super ~*cutting edge*~. I remember hearing Eric Lander talk about it in 7.012 so I was excited to actually use it. I was definitely kinda lost in the beginning but it eventually clicked, which was good. I really appreciated how we got to start from start to end--from figuring out what gene to target to actually having CRISPRi work in the E. coli cells. It felt that much more "real" in the sense that we had control over some part of the experiments. Although the whole "in silico" part and all the plasmid stuff on Benchling were super difficult and annoying to work with...

I remember Noreen telling us at the beginning of the year that when we first learn about the experimental process as young students, we think of it as a straight line, when in real life it's a huge scribble. I definitely understood that a bit better after our results showed that CRISPRi had no effect (and even worse, some conditions resulted in NEGATIVE lactate production. Like what the heck how is our data so wrong).

At least failure doesn't delay/prevent us from graduating like I presume it does in grad school... In 20.109 we can just move on with our lives and write a research article with unsuccessful results :D Also, shout out to the blue team for having working data that I could talk about in my research article

All in all, despite the Benchling struggles and my data pretty much being trash, I still enjoyed this Mod since we got to go from start to finish, which seems pretty rare in an undergraduate lab course. Plus, when I mention what I did this module to my non course-20 friends, they all recognize CRISPR from 7.012 and think it's really cool that we're doing cutting edge stuff! Thank you to all the staff who helped us through this module! Whether that was being understanding that we were all screwed for 20.320, bringing us snacks, or spending so much time explaining concepts thoroughly in lecture/lab :) Can't believe that we only have a month left to go in the semester, time really flies!

Some thoughts on engineering with CRISPR

The first time I heard about CRISPR was Fall 2014 in 7.012 during one of Eric Lander’s lectures.  He said something about how it was going to revolutionize genetic engineering, which, in retrospect, was my first wakeup call to what was happening in the field.

I didn’t realize to what extent that was true until after ~2 years of reading endless new studies and articles about how much potential this technology has.  Well, that and hearing all the terrible (and great) CRISPR jokes from every single biology professor I have had so far at MIT.  It has gotten to the point that I was sitting in next house dining a few weeks ago and I overheard someone at an adjacent table say something like “oh the fries are crisper today than normal” and it took me a solid half minute to realize that they weren’t trying to make a reference to CRISPR.  It turns out there’s this other word, ‘crisper,’ which means ‘even more crisp than an already crisp thing.’  Whoops.  I told this to a course six friend and they gave me a look as if to say ‘jeez, how far down the rabbit hole have you fallen?’  Pretty far, I’d say.  And if you listen pop sci, we’re not far off from a wonderland full of genetically engineered babies.  In reality, we’re probably not, but this didn’t temper my excitement when it turned out we were going to get to use CRISPR for mod 2!  I was ready to dive right in.  *Cue heavy-handed Corgi gif*

I really appreciated the journal club assignment, particularly because my paper dealt a lot with how CRISPR actually targets and interacts with the target DNA, which is something I had very little understanding of.  As usual, public speaking was fraught with me mumbling ends of sentences and tripping over my words, but I got through it, and, anyway, slip ups are all part of the learning process.

Most exciting was getting to see results for the ethanol assay with our ldhA targeting CRISPRi system.  And seeing that it actually worked!  Yay!  It was really exciting to get to see this technology in action, and to get to design the system ourselves.  Reading and just being immersed in CRISPR research helped me to better understand exactly how powerful the CRISPR approach is.  What better way to understand something than by testing it for yourself?

Lastly, I’d like to give a huge shout out to the teaching faculty!  You guys are amazing and make lectures and labs run so smoothly, I don’t know how you do it.  Thank you, also, for being wonderfully kind and understanding and answering all of my random questions with enthusiasm.  And thank you for the foods during journal club and the five hours I spent in office hours trying to analyze data for the research article.  This hungry, stressed course twenty student appreciates the gesture.

I am very tired now, after a long week of writing reports and taking tests (and looking for corgi gifs, as it turns out), so I am going to go sleep now, and will see you all this Tuesday!

 

The Mo' You Know

2 modules down, 1 to go. I’ve got to admit, it’s a pretty odd feeling; it seems like just yesterday Prof. Engelward was teaching us about DNA damage and comet chips, and Dr. Lyell was explaining to us the difference between CRISPR and CRISPRi (*note* - I realized this had to be a pretty important distinction given that it showed up on a Mod 2 quiz and I had exactly 0 clue how to answer the question… but don’t worry, now I know the difference). Now, here we are weeks later, having taken the first step in making phage batteries.

I think that things definitely began to pick up in this module. I don’t think it was particularly helpful that content in 20.320 simultaneously became more abstract and challenging. Definitely much more of a balancing act this time around. Apparently the 20.109 teaching staff are not crazy for constantly repeating to “start. early. on. assignments.” As they say…

In terms of Mod 2 content, I’d definitely say that my chief complaint was the abundant use of Benchling. Don’t get me wrong, I think it’s infinitely better than trying to design primers manually (spent the whole summer doing this. 0/10 would not recommend) or hand-drawing plasmids. And I certainly enjoyed getting to name the plasmids (shout out to the aristocratic Sir Victor de Cas9 Vector). But man, there’s only so long I can stare at nucleotides before my brain goes on autopilot. I swear, after a few weeks of that, I was counting plasmids instead of sheep to go to sleep.

All in all, however, I think that this module was a much better representation of what “real-world” research is like. In other words, you pour your heart and soul and time into an experiment, confident things will work correctly, and then when you finally collect your results, you realize that – well – things didn’t go quite as expected. Take our team’s data, for example (TR Green – I ain’t lying). Now, we expected D-Lactate production to be maximal under anaerobic conditions in the presence of CRISPRi and aTc. But you know what didn’t go as expected? Which condition in fact had 0 (read: negative) D-Lactate production? You guessed it. So you could say that there were some undertones of frustration and resentment in my research summary. But c’est la vie. That’s the nature of the beast. You can’t be intimidated by every setback in research. Otherwise, how would we get progress?

Philosophizing aside, I still thought this module was – in theory – pretty near. It was amazing to see how a brainstorming session could go from paper to reality. One day we were contemplating which portion of the fermentation pathway to target, and the next we were implementing our design in the lab.

Of course, none of this could have been done without our always enthusiastic and patient teaching staff! A huge thank you to Dr. Lyell and Leslie and Emily and Maxine for all of your help! 

MOAR LACTATE PLS

Mod 2 was an interesting time. I am glad that we got to use CRISPR, *the* hot topic of biological engineering. I remember, last year, when Jennifer Doudna came to give a talk at MIT and I thought the people of the Koch were preparing to go to battle. My grad student described it as a talk "too close to enemy territory." But yes, I'm happy to have finally gotten the chance to actually understand what all the excitement was about, and to use it with my own, bare [gloved] hands.

Not going to lie, but the first few days using Benchling to design our plasmids and oligos and sequencing primers gave me a literal headache. It didn't help that Michelle's laptop battery kept dying so we were down to my sad, 12.5in screen. I remember doing DNA engineering in high school, laying out the 120375 sheets of paper on a desk in front of me, and using a highlighter (with the help of ctrl+f on a computer) to design my primers. I almost prefer that to Benchling. But we made it through and we made...a thing!

Unfortunately, our system didn't work very well. In fact, it may have decreased lactate production, which is the opposite of what we wanted. But, at least someone's construct worked in our lab section, which made writing the report 100000x easier. Thank you Blue Team! I probably should have looked more into the gene and "putative" promoter region more, but time constraints existed. Writing the Mod2 report wasn't terrible in comparison to the 4 similarly-sized reports we had to do for 5.310 (that's a whole different rant for a different time).

I really enjoyed doing the Journal Club! I spoke much slower than I thought. I also put way more time into it than I thought I'd need to. But I guess that's how learning works.

Okay, that is the end of my scattered thoughts. Thanks teaching staff! You guys are the bomb! No memes from me because I'm lazy AF.

<3 Aria

'Failed' experiments...

The experimental process is quite a mess. I’ve been realizing more and more that science is imperfect and more often than not things go wrong. Unfortunately this was proven true for our Mod2 experiment. There was little to no statistical significance in the findings and this will make going home for Thanksgiving interesing since they’ll be asking what I’ve done and my response will be 'well I failed to upregulate Lactate production by using CRISPRi technology to plant a ‘roadblock’ to prevent transcription, and therefore function of the enzyme pflB to try and increase pyruvate to funnel more of that product into Lactate. And since none of the other words really make any sense they’ll just hear I failed.

                It sounds kinda sad but honestly I’m just excited to be doing real science and I wish we had time to go back and try another guide or run more trials and try and find out what went wrong since these implications are really powerful and I wish we had found something more substantial. I’m glad we got an introduction into CRISPR technology and having more of an idea of what it is amazing since it has so many possible applications. Writing the Mod2 report has been a great way to synthesize all that we learned and it opened my eyes to all the places the project could have gone wrong and so I think it was a great way to end the module! Thank you, instructors,  for all you do!

CRISPR-IRL

As I look at my calendar, I realize November is already halfway complete... where has the time gone??

I feel like I only started understanding how CRISPRi worked yesterday. That's an exaggeration. Last week. For some reason, it was really difficult to differentiate the names and roles of the different plasmids in the CRISPRi process until right before Journal Club. Even then, I felt shaky.

I was actually really excited about the Journal Club project. I've sat in on Journal Club presentations in my UROP lab meetings plenty of times, but I didn't realize exactly what was happening until this assignment. Once I realized that I would be presenting another person's work from back to front for a group of people much more intelligent than myself, I began to feel more nervous.

Despite this, I was still ready.

I was prepared to read my article several times, annotate the crap out of it, make the best figures and title slides you'd ever seen. Journal Club was gonna be totally owned by me.









And then, just like that, life gets in the way.

I couldn't find the time to put into the presentation that I'd intended. I was inadequately prepared and felt defeated before I even began my presentation.







I was sort of confused. I was probably even less prepared for the presentation I had the next day in my HASS class, yet I was not nearly as nervous about it as this one. But this makes sense. The JC presentation required much more knowledge than my HASS presentation.

In the end, this was a learning experience. I know to strictly allocate time for preparing for JC and to make sure I practice what I want to say in order to prevent my nerves from showing and taking up all of my time with the word "um."

I spoke briefly with Noreen about this, but I think it would be useful to have multiple Journal Club presentations throughout the semester. If there were more and each individual one was worth less of your grade, you could improve across the semester and not feel it was so daunting a task. We could get used to what it would be like in a real lab, doing them every week or every few weeks. If we even only did parts of it and had to present a figure to the class in a formalized structure, that practice would be so helpful in making each full presentation better.