# Module 1, Day 4

### Transformation Results

Plate Transformation Expected Results Experimental Transformation Efficiency
1 positive control plasmid most growth $\frac {~300 colonies}{5ng plasmid DNA} \frac{1000ng}{1ug} = 60,000 \frac {colonies}{ug DNA}$
2 backbone + no ligase minimal growth (neg. control) 0 colonies / ug
3 backbone + ligase minimal growth (neg. control) 0 colonies / ug
4 backbone + insert + ligase many colonies, but less than plate 1 0 colonies / ug
5 backbone + insert + ligase many colonies, but less than plate 1 0 colonies / ug

We did not observe colonies on any plates besides the positive control. This could be due to a variety of reasons, originating from problems during the annealing, ligation, transformation (less likely, because the positive control was successfully transformed), or even insert design steps. The reagents in any of these steps could have been faulty or contaminated. It is also possible that our small DNA pellets were washed away during the washing steps, or that our genomic changes in attempting to add more enzyme restriction sites with the insert resulted in lethal mutations.

### Fighting Words

"There is no such thing as a standard component, because even a standard component works differently depending on the environment. The expectation that you can type in a sequence and can predict what a circuit will do is far from reality and always will be."

Professor Frances H. Arnold, Caltech.

The above statement is certainly capable of eliciting powerful responses from synthetic biologists and other scientists who have made it their life work to develop and utilize the very standard components that Prof. Arnold pronounces "far from reality." Like any fairly reported news piece however, Pollack's purpose in his article "Custom-Made Microbes, at Your Service" was not to openly champion or disparage any particular perspective on the matter, but rather to present a fair and balanced view of synthetic biology as a new field emerging from the crossroads of more established disciplines in science and technology.

In addition to presenting vignettes of the work of current synthetic biologists, Pollack does this by quoting Professor Arnold to introduce the skepticism that synthetic biologists often face from others within the scientific community. Professor Arnold's statement asserts that unpredictable environmental conditions will make it impossible to develop biological components with consistent functionality, as is the goal of the BioBricks project.

Prof. Arnold's position may well be influenced by her own academic work, which centers around using directed evolution in the laboratory to produce useful new enzymes and biopathways. Instead of trying to design and build biological systems from the ground up by looking past the designs that nature has already given us like synthetic biologists, her lab is taking the opposite approach. Professor Arnold hopes to speed up, screen, and guide the convoluted mechanisms of evolution in a way that will give rise to new enzyme pathways that have evolved naturally (though in a laboratory setting) towards a specific purpose. Indeed, her laboratory website (http://www.che.caltech.edu/groups/fha/) describes evolution as "a powerful algorithm with proven abilit[ies] to alter enzyme function...the challenge is to collapse the time scale to months, or even weeks." She further goes on to defend directed evolution as a superior method to genetic engineering or synthetic biology by maintaining that "the relatively few examples where 'rational' design has yielded useful enzymes do not negate the fact that rational protein design is often a fruitless exercise." Though Professor Arnold's research approach is interesting, it would be easy for the nature of her work to bias her against the methods of synthetic biologists, who are tackling similar problems from a different angle.

Arnold's assertion that to be able to "type in a sequence and can predict what a circuit will do is far from reality " strikes me as easily refuted--modern day computer languages do precisely that by enabling programmers to turn high-level code into the assembly language signals spoken by circuits and transistors. Synthetic biologists are merely aspiring to parallel these achievements with DNA. Of course, "prediction" might be too ambitious of a word as perfect functionality is rarely achieved on the first try--programmers (and synthetic biologists) recognize that they must test, debug, and rework their code repeatedly before it can be released for public use. We as a society then place enough trust in these "typed sequences" to allow electronic devices and software to help us in our jobs, our health, our lives.

Even when interpreted more specifically to refer to biological systems, Arnold's quote does not hold up against achievements already made in synthetic biology. For example, many biotech companies use huge cultures of genetically altered Chinese Ovarian Hamster (CHO) cells to mass produce a variety of FDA approved medications. While these cells are not the small, general parts contained in the registry, they are biological components that can be reliably altered for a specific purpose.

Another simpler example of how biological standards can be used is when we used the M13k07 backbone as the plasmid DNA for our M13 bacterial transformations. We knew from the registry that this plasmid has been modified to include a kanamycin resistant gene, ensuring that any bacteria growing on the lb+kan plates would have been successfully transformed. Thus, we were able to put the registry to use in selecting a standard biological part (or device) that was helpful in conducting our M13 experiments.

Similarly, Professor Endy's work as detailed in the "Refactoring Bacteriophage T7" paper we discussed in class involved parts such as ribosome binding sites being duplicated and rearranged across an entire genome to produce a more understandable genomic code.

Though it is true that no two environments or organisms are ever exactly the same, these factors can and have been controlled to some degree. It is this generalized control, both in the lab and in practice, that has led to all the advances in pharmaceuticals and healthcare. Just as not all types of bricks work in all building environments and computer programs must be adapted for different operating systems, it is important part for synthetic biologists to know the conditions under which standard parts can be used. It is thus also important to not rely on standard parts in drastically new environments until they have been proven to function in the environment satisfactorily. And as with in any other field, this information is collected over time and fine-tuned as our understanding advances.

Biological agents have and will continue to surprise us by responding to their environments in unexpected ways. However, this unpredictability is not because biology is guided by some force that inherently defies understanding--biological systems adhere to the same basic chemical and physical laws as everything else in the universe. More likely, these "mysteries" behind why organisms do the things they do arise from our lack of study and understanding of the interactions between systems and their environments, and can only be elucidated through further research attempts from approaches across the board.

-Crystal