JJ Wheeler

JJ Wheeler

Jun 12, 2019

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Captain's Log #2

Written by JJ Wheeler

Hello science lovers! I’m JJ Wheeler, one of the student leads of SoundBio iGEM. As you might’ve read in the previous Captain’s Logs, our team operates in five subteams, and I am one of two leads for the wetlab subteam. Each of these logs will provide weekly updates focusing on a particular subteam, and this week I’ll tell you what wetlab has been up to for the past few months.

About Wetlab

Wetlab is the biggest of the subteams of SoundBio iGEM; it consists of 17 members, including myself and David Lu, my fellow lead, under the guidance of our advisor, Aida Hidalgo.

“Wetlab” refers to the biology laboratory experimentation side of the project; we cover any experiments with living things, chemicals, reagents, etc. The wetlab aspects of our project this year include culturing K. rhaeticus and producing bacterial cellulose (BC) (an area we work closely with the hardware subteam on), designing genetic circuits for light-controlled attachment of functional proteins to the BC, called functionalization, engineering bacteria to express these systems, and testing the functionalization process.

Progress

Culturing K. rhaeticus

One of our first experiments was testing the growth of K. rhaeticus and production of BC under different conditions. This was an important step in becoming more familiar with the organism and defining methods of culturing it in our lab, as it is necessary to inform many of hardware’s design decisions.

The wetlab team met to deliberate over what we wanted to learn from these initial growth experiments, and we brainstormed a list of several culture conditions we wanted to test: agitation level, container volume/type, carbon source, media supplements, temperature, aeration, and co-cultures with E. coli, as we are exploring the idea of growing the BC and functionalizing it all in one culture by co-culturing K. rhaeticus with our genetically engineered E. coli. Out of these various conditions, we decided that the most immediately important and simple conditions to test would be agitation and the container type/culture volume. These two factors are essential in determining basic elements of our bioreactor design. Agitation is especially important, as the level of agitation will affect how the BC forms in the culture; we need to find the right agitation where the BC membrane isn’t substantially disrupted as it forms, rendering functionalization impossible, and where oxygen delivery is still maximized, as oxygen is essential to BC production.

For this season's first wetlab experiments, we set up an array of cultures to test how BC grows in different containers and with agitation. Pictured below is our collection of cultures of various volumes in different containers.

The containers we tested were 100 mL  Pyrex bottles, culture tubes, 50-mL conical tubes (the yellow ones have vented caps made for letting air in, the green ones have plain caps), and petri dishes. We tested petri dishes with agitation and without agitation; as our orbital shaker was broken, we had to tape them up to a vortexer to get some agitation. Each day, observations on the thickness, texture, shape, color, etc. of the cellulose of each culture are recorded.


Genetic Circuit Design

A major part of our work so far has involved identifying the genetic parts necessary for light-controlled protein expression and functionalization of BC. For now, we are planning to work with well-characterized Biobrick parts used by previous iGEM teams. Biobricks are standardized DNA parts available in a Registry for constructing synthetic biological circuits. Our genetic circuits will consist of three main elements: a light sensor protein, a regulator controlled by the light sensor protein and the promoter it acts on, and a coding sequence for a fusion protein consisting of a cellulose binding domain (CBD) and a chromoprotein placed under control of the regulator. Pictured below are our first genetic circuit designs.

Figure 1: Cph8 red light system. Cph8 is the red light sensor. Cph8 affects the activity of OmpR, which regulates expression of the sequence under the promoter ompC. The coding sequence for OmpR is not present in the circuit as OmpR is already present in the E. coli genome.

Figure 2: YF1-FixJ-FixK2 blue light system. YF1 senses blue light and affects the activity of FixJ, which regulates expression of the sequence under the promoter FixK2. This system works in the same way as The constitutive promoters, terminators, and ribosome binding sites of the blue light system will be the same ones used in the red light system.


For now, we have chosen two different light sensing mechanisms to control the functionalization of the BC: the YF1/FixJ blue light sensor, and the Cph8 red light sensor. Both YF1/FixJ and Cph8 are well characterized and have been used by multiple past iGEM teams, notably Team Exeter in 2013. These systems can turn gene expression off when cells are stimulated with the correct wavelengths of light, allowing us to target where we don’t want our engineered bacteria to functionalize the BC--in other words, it allows us to target where we want to attach proteins of interest by not shining a certain wavelength of light on that spot. We’re interested in showing control over concurrent and independent functionalization with at least two proteins, so we wanted to test at least two light sensing systems.

Cellulose binding domains (CBDs) are small proteins we will be using to attach our proteins of interest to the BC. Although there are many methods of attaching proteins to a substrate, we have chosen to focus on CBDs as this method was explored by Imperial College iGEM in 2014 and we will be able to build upon their work. For now, we have chosen to experiment with the CBDs characterized by Imperial College: dCBD, CBDcex, CBDclos, CBDcenA, and CBDcipA. These CBDs are linked to the protein of interest to create a fusion protein. The expression of the protein can be controlled by the aforementioned light sensing systems.

While our project’s overarching goal is to create a platform for functionalizing BC with a protein of interest for any given application, we want to work on first attaching visible chromoproteins to the BC as a proof of concept. We have chosen one chromoprotein to work with, superfolder green fluorescent protein (sfGFP), and are currently in the process of identifying additional chromoproteins to work with. We chose sfGFP because it was also used by Imperial College iGEM 2014, so it is well characterized and fusion proteins containing sfGFP linked to various CBDs listed above are already designed and in the Biobrick registry.

We have recently started transforming the fusion protein and optogenetic parts discussed above from the iGEM DNA distribution kit into E. coli. A bacterial transformation is the process of manipulating cell walls to make them more likely to take up foreign plasmid DNA. This step will make each individual part readily available for the cloning work necessary to construct our full genetic circuits. We are currently in the process of finalizing the designs of our genetic circuits.

Future Directions

In the near future, we are going to continue observing K. rhaeticus growth. Additionally, we will likely coordinate with the hardware team to set up additional growth experiments under different conditions, such as tests with different media supplements, carbon sources, and aeration levels.

We will also continue work on our genetic circuit designs. We plan to have our initial designs released in the near future, before our July 1 “Plasmid Design” project milestone, and in time for the next deadline for free synthesis offered to iGEM teams by Twist Bioscience.

Finally, we are continuing to review literature on various subjects related to our project. As of now, our major areas of research are: i) CBDs and other protein attachment mechanisms, so we can determine the most efficient ways of attaching functional proteins to BC; ii) optogenetics and light-sensing mechanisms, so we can better understand the genetics of light-controlled expression and make informed decisions on genetic circuit design and how to target functionalization of BC; iii) co-culturing K. rhaeticus (mainly with E. coli); and iv)  protein secretion in E. coli, as we need to better understand the mechanisms by which our genetically engineered organisms will release the functional proteins so they can attach to the BC.

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About This Project

The bacterium Komagataeibacter rhaeticus has the ability to naturally produce bacterial cellulose (BC) which possesses many unique, highly useful properties suitable for a wide range of applications. We hypothesize that an optogenetic circuit in an engineered strain of K. rhaeticus grown in an optimized bioreactor can spatially control attachment of proteins to the surface of BC membranes to enable fine-tuning of these properties for different applications.

Blast off!

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