Berkeley Engineering Blogs

This proverb seems to hold true when referring both to our short-lived success last week and to the official conclusion of full-time work for iGEM. As for the actual status of our lab work, all of the undergrads are planning to continue our work on the project in between classes and other commitments during the semester. As Bing mentioned in his post, it turns out that last week’s exciting results aren’t reproducible and we don’t have a sound promoter after all. The fabA promoter which appeared to be induced by audible sound is actually induced by something else–when the audible sound and ultrasound experiments were repeated multiple times, the control was “turned on” in some instances and not others with the same experimental conditions. Additionally, when Christie sequenced the construct with the Green Fluorescent Protein (GFP) under the control of what she thought was the fabA promoter, she found that it was actually the PfhuA promoter (an iron-dependent promoter) preceding GFP. Even when she tested the actual fabA promoter and the PfhuA promoter, she saw mixed activity in the experimental control on different occasions.

The results in testing the sound promoters are pretty much inconclusive, and we are now refocusing our project around some of the applications of sound-induced lysis that appear like they will work–Gateway and composite part Assembly. So, we are currently investigating three schemes for making a Lefty-Righty Gateway reaction work in vivo, or in the living E. coli, while minimizing the background of unwanted projects that we get. In previous experiments, I have shown that this reaction does work under the conditions in the cytoplasm of cells that express the necessary proteins (xis, int, ihfA, and ihfB). One of the schemes involves selection against the undesired products by utilizing the lethal gene ccdB. The other two schemes involve positive selection for the desired products using conditional origins of replication (which are only active in the presence of specific proteins) or plasmids that can be packaged in phages.

As for assembly, we are currently making both cytoplasmic and periplasmic variants of BamHI-ligase and BglII-Cre parts. Both sets of parts are targeted at performing assembly in the lysate since each of the two cultures coming together contain two of the four required enzymes as well as one of the plasmid substrates. Since Bing does have a few variants of the lysis device, we will be using one that is arabinose induced (with the lysis device under the control of the pBad promoter) once we bring the two cultures together. Our experiments show that the assembly reaction does not happen in the LB (or the “Lysogeny broth”-the nutritionally rich medium in which we grow our cultures), but the reaction should work well if the cells are resuspended in buffer before lysis.

Chris has also been doing a few experiments along the lines of assembly within the cytoplasm. He has found that ligase at medium copy is stable in cells. He also has found that ligase is able to bring together sticky ends from restriction digests before DNA polymerase can fill in the single stranded region. These findings have led him to consider using plasmids methylated at either the BamHI or BglII sites (depending on whether the parts contained are intended to be on the right side or the left side of the assembly), which will be digested with BglII, BamHI, and XhoI in the presence of ligase. The resulting mixture can be transformed into a strain expressing ligase (with the ligase gene integrated into the genome). The idea is that the BglII-BamHI scar sequence will be stable in the presence of the restriction enzymes, so only the XhoI site will remain digested in the desired product. After transformation, the ligase in the cell can then ligate at the XhoI site to yield the desired product, which can then be isolated from the cell.

In addition, Aron has been able to test some of the prepros to see that they work, and Sherine and Cici have been working on making and testing the tag sequences. With that final note, I have pretty much summarized where the majority of our lab work stands at the moment.

Switching gears a bit from the technical side of things, I can’t believe that the summer (and with it, full time work for iGEM) has come to an end. It has been quite an experience working with such a talented and motivated set of people. Despite all on the long days (and nights) spent working in Stanley Hall, it has been a very enjoyable experience (particularly when we did take the time to do fun things as a group and realized that we couldn’t get our minds completely off lab things). I think that our ability to work through all of our setbacks and problems, both small and large, has made the experience all the more rewarding and helped us bond with each other. Even now, as we embark on shifting the project focus and actually completing the cloning steps to implement our proposed schemes, I can appreciate the volume of things that we actually have done over the past few months. By virtue of us exploring work along so many fronts, we have been able to traverse many paths though many of them have unfortunately led to dead ends (which seems all too common in science). In any case, hopefully we will be successful in at least one of our ventures with continued hard work and a bit of good luck.

I’d like to wrap up with a few pictures of what we do in our spare time:

Apparently, we were looking for something historical when we visited the San Francisco Museum of Modert Art.

Trying to fit in at SFMOMA...

The so-called Jinja.

Bing, have you been spending too many nights in the lab?