Background

While some bacterial infections are seen as common place, others remain elusive to cure. Tuberculosis has currently infected a third of the world’s population, and 1.7 million people died from it in 2009. As shown, it is a worldwide problem that is prevalent in third world countries.

Tuberculosis exists in two stages. In its latent stage, the immune system has it under control and prevents the bacteria from reproducing. When it switches into its active stage as a person's immune system becomes compromised, it begins to multiply and the person starts showing symptoms of the disease. The genetic mechanisms that trigger the change between the two forms are not very well understood. The genetic network of tuberculosis is complicated and studying the interactions between genes can be time consuming, requiring the construction of many plasmids to study only one small part of the network. To look for new, faster ways of studying this problem we turned to synthetic biology. Instead of creating many vectors to study the interactions of genes, we aimed to create a plasmid with a designed circuit that would control the transcription of several tuberculosis genes using invertases. We chose to use non-pathogenic mycobacterium tuberculosis genes and transcription factors within non- pathogenic E.Coli for safety reasons.

Invertases are recombinases that will bind to recognition sites. Once bound, the DNA between two sites will invert and flip horizontally. This effectively acts as an on/off switch for transcription of that region of DNA as it is not available for DNA polymerase to bind to. In terms of circuit design, it will act as a not gate. Using them within a plasmid with several tuberculosis genes would allow us to check for regulatory measures such as negative feedback, etc.

An example of a construct built around the invertase architecture.

In addition to the novel cellular architecture, we also used a variety of software tools created by our computational teams (Boston University, Wellesley):

• G-nome Surfer Pro
  G-nome Surfer Pro is an integrated environment that allows for the viewing of prokaryotic genomic data and literature associated with the genome. As it is built on a Microsoft Surface, it encourages collaboration. We used the Primer Genie to design the primers for the tuberculosis genes and transcription factors.
• Trumpet
  Trumpet consists of a library of genes and promoters, which can be configured into any desired permutation or combination by treating the DNA with recombinases, which allows us to rewire the genes and switches and study all their combinations. It was used by the wetlab to help us map out where the invertases should go in our plasmids in order to correctly turn on and off the segments of DNA we want to study.
• PuppetShow
  This suite includes a high level programming language for specifying biological protocols commonly used in the laboratory, which are then executed by a liquid-handling robot with minimal user intervention. We used this in conjunction with the robot (described below) to create samples that were simultaneously created manually to compare the results.
• Clotho
  Clotho is used to mange, create and store new biological building blocks in community based repositories. It includes a suite of tools that include PuppetShow and Trumpet, which were designed specifically for this project. Other tools that were used were:
  • Bull Trowel
  • Spreadit Parts
  • Spreadit Vectors
  • Spreadit Features
  These were used to store a variety of parts, features, and allowed us to assemble vectors we were interested in creating electronically.
  • Optimus Primer-primer designer
  • Feature Chomp-reads in APE and GENBANK files and takes the features found within the file to a feature database
  • Batterboard-allowed us to electronically represent physical samples in the lab

Another way we looked into facilitating progress in studying genetic networks is through the use of automation:

• Robot
The liquid handling robot was used to reproduce molecular biology protocols in a more efficient and accurate manner. The robot is controlled by Puppeteer which creates code necessary to choose the correct series of commands in EVOware,the robot's software.
• QIA Cube

Through the usage of novel cell architecture, software, and hardware we show that studying complex genetic networks can be done faster than has been done in the past. It reduces the amount of work needed to be done, as well as the demand on materials and time.

Results

Being associated with a large computational team, the wetlab team was unique as our results not only consisted of creating novel forms of DNA, but evaluating the usefulness of the software tools our computational team produced. Our success was not only measured in the number of colonies we could get to grow, but also in how well we could incorporate these tools and give informative feedback.

Clotho: Key to Creation

Clotho allows one to enter a variety of forms of pieces of DNA including parts, features, and backbones into a database. To do so, one uses BullTrowel, Spreadit Vectors, Spreadit Features, and Spreadit Parts to create a representation of the DNA for the database. We entered promoters, RBS, genes, an terminators as parts and the backbones as the plasmids. We could then create vectors of all these pieces of DNA that we would then want to go physically construct in the lab. A feature is any piece of DNA (part,backbone,etc.)that one may want to highlight in a long piece of DNA. This would allow us to highlight things such as restriction enzyme sites in the vector.

The first vectors we were interested in creating were fluorescent protein reporters. Our goal was to eventually fuse genes with the reporter devices in order to establish that the genes were being turned on or off, but we first wanted to characterize the luminescence of the reporting devices. We used Clotho to store the pieces of DNA we were hoping to create, listed here:

Parts and diagrams.

This list of parts would give us red, green, yellow, and blue colors to play with. We then set out to create the devices.

Automated Creation: It was the best of the times; it was the worst of times

Using Puppeteer, the BioBrick construction process was automated. Ligation and restriction digest could be done using...
It is important to understand the behavior of each promoter before using it in a complex plasmid architecture, because a baseline is needed to establish how a promoter behave independently of any additional transcription factor. In the wet lab, various devices were created with either constitutive or inducible promoters. The constitutive promoters like Bba_R2000, Bba_I14033, and Bba_R0040 cause the genetic devices to produce fluorescent protein at all times. But the inducible promoters like Bba_I13453 need arabinose in order to promote the transcription of the fluorescent protein gene downward. At first, we encountered a problem involving the failure to induce the pBad promoter. After adding various concentration of arabinose and using different protocols, it turned out that the commercial cell lines, which we used to solve the ligation problem, did not contain the AraC gene. Since the AraC gene has to exist and interact with arabinose to activate the pBad promoter, we then looked up the part that contains AraC gene and added it to the existing part. While no fluorescence was been observed under UV, the analysis of those samples with the FACS machine has produced an arabinose concentration versus fluorescence intensity data that showed significant increase in fluorescence intensity after the induction with arabinose.

Genome Surfer: Surfing like a shark, hitting like TB

Optimus Primer: More than meets the eye

After we characterized the promoters with the FACS machine and researched the TB genes using Genome Surfer, we could theoretically start building a genetic device with a TB gene fused into the fluorescent protein gene. But we still had to design a primer that can amplify the TB gene and attach it with restriction sites during a PCR reaction. This was where Optimus Primer played it major role. Because this application is integrated with Clotho, a selected sequence from the TB gene can be crosschecked with the feature list so that the amplified sequence did not contain any restriction site. By clicking the “Generate Primer”, I had this program to produce 6 primers for both the forward and reverse orientations. For each set of the 2 primers (FF and FR), Optimus Primer calculated the differences in length, the melting temperature, and the free energy. In case possible dimerization is detected, the bottom panel will turn pink. Because specific insert can be added into the 5’ and 3’ ends of the planned primer, the restriction sites E & X and S & P are added into the 2 respective ends of the primers. This step is taken because the amplified sequence needed to be combined into a genetic device for the construction of complex plasmid architecture. After the planning was completed using Optimus Primer, the provided primer sequence was ordered. The primer was then used in a PCR reaction.

Trumpet: We know all the tricks

Approach

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