Berkeley Engineering Blogs

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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?

South Dakota’s POET, one of the largest corn ethanol producing companies in the world, announced last Tuesday “that construction will be completed on a $4 million pilot-scale cellulosic ethanol production facility later this year.”   The announcement is rather vague on what defines “cellulosic ethanol,” instead focusing on the great leaps that POET has made in recent months in company labs, which its leaders see as stable enough to form the necessary foundation for “the commercialization of cellulosic ethanol.”  The company is coining this commercialization as “Project LIBERTY” (their emphasis), and, as the name would imply, the focus is almost entirely on the issue of national energy security.  In their words:

“More than 65 percent of the petroleum used in the United States comes from foreign countries.  And our dependency is growing.  As a domestic, renewable source of energy, ethanol has proven to reduce our dependence on foreign oil and increase our nation’s ability to control its own security and economic future.”

Embedded in this statement are many assumptions the company is imposing on their proposed consumer audience, for purposes of marketing their product:
-It is an American right to consume energy in the amounts we do, and access to independent transportation is a right not a privilege.  Thus, we have to find a solution to find enough fuel to support our energy need.
-Any American energy need is disentangled from a global energy need—think nationally, not globally (and certainly not locally).  However, removing the context of the global energy situation is unwise, especially for Americans, as according to the American Almanac, and a statistic which is cited frequently, “Americans consume 26 percent of the world’s energy,” even though we account only for 5 percent of the world’s population.
-Reducing dependence on other countries supersedes reducing our dependence on energy in general.

Cellulosic ethanol, we are told, is distinguished from regular corn ethanol here by the fact that it is made by breaking down the rougher parts of the corn plant–and for POET specifically, the corn cobs.  However, according to research done by Jay Keasling’s lab at JBEI, the technology is just not there yet for breaking down the complex structure of cellulose and lignin to turn it into sugar.  Here we run into the curious interface between corporate and academic sectors, especially when thinking about the open-source nature of synthetic biology associated with Keasling and the profit-driven sector in which his product will exist.

Some questions I have:
-How are habits changed?  The global situation of energy consumption and global warming could conceivably be a large problem requiring large change.  How will habits associated with consumption, lifestyle, and usage of infrastructure be impacted?  From where or whom will the impetus for such change come?
-For many biofuels producing companies, there is a focus on using the current extensive infrastructure surrounding independent transportation (gas stations, fuel lines, etc.).  What is the threshold for abandoning an infrastructure when it is proven to be inefficient?  How might it be reconstructed to allow for less wide-scale waste?  Is there space to find a more efficient infrastructure?
-Who has the power to change standards and how?  How is the right amount of change found?

Yesterday’s audible sound test ended in bright green glowing results!  Glowing means that the gene which encodes Green Fluorescent Protein was initiated by the sound promoter, which is upstream of the gene.  Meaning one of the sound promoters works, at least in this construction.  The experiment will be done one more time (this being the third test) on an extremely tight ship just to double check that all inconsistencies are eradicated.  Then the sound promoters will be assembled with Jin’s lysing part and the extremely important testing of killing cells by yelling at them will begin…

The team used a scorpion-detecting flashlight, which shines UV light, to get the samples to glow.  The tube on the right contains a pellet of DNA that was not exposed to audible sound:

Jin was extremely pleased:

And Christie did a victory dance:

Sound check number 2 happened today!  Looking for sound promoters that are attached to GFP (Green Fluorescent Protein), means first exposing the cultures to ultrasound and regular sound (in the form of “white noise”), then centrifuging down to pellets that can be exposed to ultraviolet.  If they glow green then it worked and we’ve found our parts!  (for sound promotion..)

It looks a little like this:

Two tubes hang out as ultrasound is blasted through them.  Christie says:  “Glow, cells, glow!”

The elaborate sound system with water proof ear buds blasting white noise at some cells.

Blast blast blast.

Chris is curious about the set up.  “Aren’t the headphones going to be destroyed…?”

And last but not least:  pipetting adventures result in encrypting messages for fellow igemmers–

We’re hopeful about having finally found promoters!

This is what synthetic biology is all about!

On Wednesday, the UC Berkeley iGEM team met up with the UCSF iGEM team and JBEI’s high school biotech group in Golden Gate Park for a day of BBQ.  The UCSF iGEM team is made up mostly of high school students, and their project is also focused on foundational technologies.  The high school biotech group is looking for enzymes that eat cellulose and produce cellulase to help the greater goal of JBEI’s biofuels program.

It was a great day away from the grueling lab work building composite parts…

Chris explains how he wanted to be a graphic designer when he grew up.

The team enjoyed a hearty game of capture the flag, although there does seem to be an awful lot of standing around.

Jin asks:  Do you want some cheesy dogs?  I made them myself…