Dave Gray's Build-A-Gene Experience Notes

These are some notes regarding the Build-A-Gene class experience. More technical class notes can be found here.

Motivation

I was thrilled to here that the class was available. The thought that I could have the experience of artificially introducing a gene into a living organism during my lifetime without taking a college course in advanced biology was amazing. It's not that I had a wierd urge to make bacteria fluoresce under UV light. But genetics is up there with the theory of relativity and quantum mechanics in offering a peek into how the universe works, and this was an opportunity to get hands-on experience which should massively increase my understanding of this important field.

The Instructor

Lisa Scheifele, a professor at Loyola is conducting the course. She is doing a fine job. Clearly, she has spent quite a bit of time in preparation for each class, ensuring we had the right materials and notes to complete each successive session. The process has many steps and each one is a learning experience. Lisa has also made herself available via email to answer questions that we don't think to ask during class, and I will include links to my emails and her responses below.

The Sessions

In each session, Lisa provides us with written instructions describing what to do, coaches us as needed and answers questions that we raise. The process involves a lot of, as she says, moving around tiny bits of clear liquid. That's because the DNA is so invisible to the naked eye and are the other molecules we work with. The results can be seen at several steps as we use a procedure called gel electrophoresis to sort out the DNA strands by size. This allows us to see if they are as long as expected.

In Session 1, we covered the basics of using the pipettes and tiny plastic "test tubes" (about the size of a pen cap). We learned about a process called "PCR" which allows DNA to be rapidly replicated into a very large number of copies. We then followed instructions to copy some DNA components that we will be working with. You can read the instructions for that session here. Afterwards, I sent an email with some questions for Lisa. My questions and here responses are here.

In Session 2, we used gel electrophoresis to check the results of our PCR from session 1. (My results were not stellar, but still something I could work with. This was my first time, after all!) We also used a technique called PCA to assemble short lengths of artificially produced DNA into a complete strand. Then we used PCR to create many copies of the fully assembled strands. (The process actually leaves a lot of incomplete fragments, but the way PCR works ensures that only the complete strands get copied. By the time PCR is done, there are many more complete strands than fragments.). My emailed questions to Lisa after Session 2 and her answers are here.

In Session 3, we used gel electrophoresis to check the results of our PCA/PCR in Session 2. (My results were zilch, but no matter. It's a learning experience!) In this case, we mixed our own gel for electrophoresis, so that was interesting. Next, we purified our DNA from sessions 1 and 2 to remove everything other than the DNA itself, added restriction enzymes to snip off the sections we want to join and put them into an incubator. (There they float in foam in a hot water bath.) I sent a few questions to Lisa after this session which we discussed in class. My questions and notes regarding her responses are found here.

In Session 4, we combined the segments of DNA that had been cut with restriction enzymes in Session 3 so that their "sticky ends" could bond, forming a complete loop. We added T4 ligase and a buffer so that the ligase could "zip up" the DNA backbones at the join points forming more stable covalent bonds rather than just the hydrogen bonds caused by the attraction of the nucleotides.

Next we did "transformation", causing some of the bacteria to accept one of our DNA loops. This involved adding TSS (Transformation and Storage Solution) to three vials containing the bacteria and then separately mixing in water (a negative control) the positive control and our ligation, each to one of the vials. These are left to cool on ice for half an hour, then subjected to a "heat shock" for 30 seconds. This encourages the bacteria to suck in our little loop of DNA. Then we incubate for an hour allowing the bacteria to recover from the shock.

Next, we spread some of each of our three products on separate petri dishes containing a layer of agar (a growth medium for bacteria) but also containing an antibiotic. After putting a large drop of the bacteria mixture on the agar, we used small glass beads to spread the bacteria across the surface, rolling them back and forth. We expect that the bacteria mixed with water will all die since they will not be resistant to the antibiotic. We expect that some with the positive control will multiply since this provides them with resistance to the antibiotic. If either of those outcomes do not happen, this means there was a problem with our technique. We hope that some mixed with our ligation will also multiply because of taking in our loop of DNA which contains a gene providing antibiotic resistance. These were left to work in the incubator.

A fun fact I learned - You should label the BOTTOM of the petri dish to identify its contents. If you were to label the tops but then drop the petri dishes and the lids went rolling, you would have no way to know which lid goes with which base.

During some of our "wait" time, Lisa conducted some interesting discussions about why specific restriction enzymes were used and ways to prevent matches to DNA locations that you don't want cut. She discussed the philosophy of the Biobrick vector and let us know she is hoping to contribute our emGFP gene to the parts registry at partsregistry.org. She also told us about Craig Venter's work, creating a synthetic genome, the Gibson assembly process and his well know statement that DNA is the only software that creates its own hardware.

In Session 5, we performed "colony PCR" in order to produce many copies of our gene from a bacteria sample that survived in the petri dish, thus indicating it had taken on our vector with that provided resistance to the antibiotic. "Colony PCR" begins with heating the bacteria to the point they burst so that we can amplify the gene from our vector. Next, we used gel electrophoresis to check the size of the resulting DNA since we only want copies that include the complete gene.

Next we discussed how we could sequence the gene in our bacteria using a technique called "Sanger", "Chain Termination" or "Cycle" sequencing. This technique can sequence up to about 400 base pairs in any one direction. Since we can sequence from both ends of our gene, we can handle 750 base pairs with some overlap in the area that is less reliable (the center fo the gene.) As long as either sequence found a match for the expected nucleotide in that area, we would assume the sequence is correct.

We also checked out some websites that show how sequencing results look, help you to compare your results to what you expected and also to reverse the sequence for comparing the results of sequencing from the other end.

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