Accessibility

Introduction

We believe SMORE should be accessible to every researcher who needs it and everyone who likes to learn about it. An accessibility regardless of academic background is important to innovation through interdisciplinary research, a notion we explored in the Interdisciplinarity page.

Nonetheless, an exchange of skillset and knowledge can be difficult. Technical language forms communication barriers. A steep learning curve hinders the gain of new skills. How can we make SMORE an accessible platform that promotes interdisciplinarity?

We improved the accessibility of SMORE in 4 aspects: readability, hardware, user experience and data. These help our project to become easier to understand to use, as a step to diversify the field of synthetic biology.

Readability

To provide everyone with the opportunity to understand SMORE, our first step was to increase the readability of our wiki.

Introduction is where most readers start from. And technical details in later paragraphs are often reserved for the experienced and the interested. Therefore, we write introductory paragraphs on main pages in a highly readable language, as they are targeted to a wide audience.

The readability of the introductions was carefully set to a 12th Grade student level. This is because the iGEM community includes high-school students. And thus, synthetic biology topics should be available and understood by a high-school level audience.

To obtain an estimate on readability, we chose the Dale-Chall formula over other readability indices [1]. Unlike other indices, result of the Dale-Chall formula does not depend on the number of syllables. Many scientific terms with a high syllable count has no short and accurate substitutes. For example, “significance” has many syllables, but it is irreplaceable in scientific statistics. Moreover, our skill exchange survey showed that technical language, when used moderately and precisely, also helps understanding. Dale-Chall formula does not weigh heavily on syllables, and directly measures the difficulty of words. This encourages us to explain the difficult words, rather than to use inaccurate substitutes.

Dale-Chall formula: $0.1579*(\frac{difficult\;words}{words})*100+0.0496*(\frac{words}{sentences})$

The Dale-Chall formula includes a collection of non-difficult words. We used the updated word collection to calculate the readability, using an online calculator [2-3]. The score of the index can be converted to grade level (Table 1). Familiar jargons, i.e. SMORE, iGEM, DNA and other repeating names, are excluded, given they have been thoroughly introduced. A simple word in the collection is used to replace them in calculation.

Below is an example of readability change in our project description. We have referred to simple guides on writing readable sentences [4-5], but we also developed some tricks through the process. The change was subtle and perhaps hardly noticeable. But it greatly benefits the reader in a long passage, as proven by our own reading experience.

Original text (readability = college graduate):

Introduction

Introduction

Introduction

Introduction

Symbol	Description

Overview

Material needed

1. We cloned Dre, VCre, SCre, and Vika generators into Biobrick format, removing all illegal sites when necessary.
2. We assembled T7-LacO-Cre generator and cloned it into Biobrick format.
3. We successfully assembled 12 out of 15 of measurement constructs to allow users to quantify recombinase activity in vivo.
4. We assembled 10 target sites for Cre and proved their functionality in vitro. We also assembled the target sites for Dre, VCre, SCre, and Vika – Rox, VLox, SLox, and Vox respectively. Their functionality is proved in the measurement constructs.
5. We have extensively quantified the recombination efficiency of the five recombinases in E. coli.
6. We have built software and used it to design assembly methods for six logic gates using tyrosine recombinase.
7. We have built deterministic and stochastic modeling to simulate the behavior of site-specific recombinase. We also devised an algorithm to detect potential recombination sites in a genome.
8. We have conducted an investigation into interdisciplinarity. This includes a survey to identify challenges in interdisciplinary work, and a systematic analysis of past iGEM teams, to test correlation between interdisciplinarity and iGEM achievement.
9. We have integrated the result from the interdisciplinarity study to improve accessibility of SMORE in four aspects: readability, hardware, user experience and data.

Cloning Dre, VCre, SCre, Vika

Using PCR mutagenesis, we have successfully removed the illegal XbaI site from all four recombinases.

We then cloned the T7-LacO-regulated recombinases into biobrick format. For SCre, there are two illegal PstI sites within the coding sequence (CDS), and for VCre, there is one illegal PstI site within the CDS. We have successfully removed all the illegal sites.

We submitted all four recombinases as T7-LacO-regulated generator, in pSB1C3 and contain no illegal sites (BBa_K2406081, BBa_K2406082, BBa_K2406083, BBa_K2406084).

T7-LacO-regulated Cre generator

We have successfully cloned T7-LacO out of pET28b, and used the cloned fragment to perform a five-part MoClo assembly. The assembled T7-LacO-regulated Cre generator was then cloned into biobrick format (BBa_K2406080).

Standardized measurement constructs

We have created standardized measurement constructs to quantify recombinase activity in vivo. They are essentially transcriptional terminator flanked by two recombination target sites, inserted between a constitutive promoter and a RFP gene. Of fifteen possible combinations for five recombinases (Cre, Dre, VCre, SCre, Vika), we have successfully generated, sequenced, and submitted twelve of them:

Assembling and testing the 14 recombination sites

We have successfully cloned the Rox (BBa_K2406000), Vox (BBa_K2406001), VLox (BBa_K2406002), and SLox (BBa_K2406003) in to pSB1C3 biobrick for use. Their functionality is demonstrated in the measurement constructs, described above.

We have also cloned ten additional target sites that can be recognized by the Cre recombinase. They are called Lox511 (BBa_K2406008), Lox2272 (BBa_K2406009), Lox5161 (BBa_K2406010), LoxN (BBa_K2406011), M2 (BBa_K2406012), M3 (BBa_K2406013), M7 (BBa_K2406014), M11 (BBa_K2406015), Nuoya (BBa_K2406016), and Zsoka (BBa_K2406017). They are all proved to be functional by in vitro assay using cell lysate containing Cre recombinase.

Quantitative measurement of recombinase activity

We have co-transformed both of our T7-LacO-recombinase generator and our measurement construct into E. coli BL21 (DE3). These strains are then incubated with or without IPTG, on either LB plate over 48 hours, or in LB media in plate reader. We have thoroughly characterized the recombination efficiency of the five recombinases, and determined what combinations are the most orthogonal pairs for future applications.

We have determined that [probably] Dre/Rox recombinase is the most efficient SSR in E. coli, and that it is orthogonal to the rest of the recombinases. However, [describe what pairs are not orthogonal]. Therefore, we recommend using __ and __ for parallel reactions in E. coli.

Logic gates and software

We have designed two-input OR, NOR, AND, NAND, XOR, and XNOR gates using the excision property of two orthogonal tyrosine recombinases:

As they contain a high degree of repetitiveness, we had difficulty ordering them as single DNA. Therefore, we designed software to ______

Modeling the behavior of site-specific recombinase

We have built deterministic and stochastic models to simulate the behavior of our E. coli strain used for measurement (BL21 (DE3) E. coli carrying T7-LacO-recombinase generator and measurement constructs). The model is able to predict that the leaky expression of recombinase can induce a significant degree of terminator excision, leading to a moderate background expression of RFP.

Furthermore, we have developed an algorithm and used it to scan through the genome of E. coli BL21 (DE3) strain, and identified five genomic regions that may potentially be a functional target site for Cre recombinase.

Human practice: Interdisciplinarity in Synthetic Biology

Due to the interdisciplinary nature of SMORE, we investigated interdisciplinarity in biology to understand how people would use it.

We conducted a skill exchange survey with Team Bulgaria and Israel. The survey identified the use of technical language as pivotal in mutual understanding in interdisciplinary collaboration.

We also measured the diversity in discipline of past iGEM teams and analyzed it with iGEM achievements. We found no significant correlation between diversity and achievements. We proposed hypotheses to explain the result.

Human practice: Accessibility Improvement of SMORE

From the aforementioned study, we identified challenges in interdisciplinary work and decided an improvement of accessibility is needed to promote interdisciplinary use of SMORE. We improved accessibility in four aspects:

Readability: we wrote highly readable introductory paragraphs for a wide audience. We also provided highly readable protocols for the software and the cell sorter.

Hardware: we devised a microfluidic device with a 3D syringe pump – an alternative to the expensive cell sorter in the market – to use with SMORE’s randomizer strategy.

Software: we wrote an oligonucleotide designer programme to help the inexperienced to design oligos to use with SMORE.

Data: we compiled and experimentally verified recombinase-related sequence data to establish recombination as a convenient and reliable technology.