"

From 2010.igem.org

Contents 1 Project Abstract 2 Project Details 2.1 2010 Illinois iGEM Bioware Team 2.2 Project Proposal: BioAlchemy 2.3 Bacterial Decoder and the Registry of Standard Parts 2.4 Synthetic Gene Networks in Synthetic Biology 2.5 Cell Surface Engineering 2.6 Bioethics and Human Practices in Synthetic Biology 3 Protocols 3.1 Making Electro-Competent Cells 3.2 Site Directed Mutagenesis 4 Results

Project Abstract

The 2010 Illinois iGEM Bioware team project has two main components: development of bacteria capable of bioremediation and refinement of a bacterial decoder developed by the 2009 Illinois iGEM team.

The bioremediation portion of our project will focus specifically on heavy metals. To achieve our goal of complete removal of harmful heavy metals, we plan to introduce genes into E.coli that will make the bacteria resistant to the metals being removed, and also genes that code for metal binding proteins that will be displayed on the bacteria’s outer membrane.

The bacterial decoder portion will be implemented using small RNAs and regulatory proteins to regulate the assembly of protein products unique to a certain set of inputs. This regulation under unique, user specified environmental circumstances is central to making the decoder a novel concept. The system implemented by the decoder will consist of different types of logic gates. These will be submitted to the Registry of Standard Biological Parts to be used by other synthetic biologists.

Our ultimate goal is to incorporate our bioremediation project and our bacterial decoder so that bacteria will be able to isolate specific metals based on their environmental conditions.

Project Details

2010 Illinois iGEM Bioware Team

Project Proposal: BioAlchemy

This year, the Illinois iGEM Bioware project will serve iGEM and Synthetic Biology in 4 facets: 1.) As the success of Synthetic Biology and iGEM is contingent upon the ability to build upon and reuse previous iGEM projects and parts, the Illinois iGEM team will be continuing the work of last year’s project: The Bacterial Decoder, while intending to incorporate additional parts from the Registry of Standard Parts into fine-tuning our final construct, 2.) As the doctrine of Synthetic Biology hopes to successfully create and modulate basic genetic regulation to perform human-defined functions, the Illinois iGEM team will continue to explore both transcriptional and post-transcriptional regulation as candidates for bi-stable genetic switches to be implemented into the Bacterial Decoder, 3.) In future hopes to create and modify biological systems to solve tomorrows problems today- in health, medicine, energy, environment, industry, etc., the Illinois iGEM team will use the metal-respiring and metal-detoxification systems in microbes to create a biological system that functions as a bioremediator which isolates and congregates toxic metals found in its environment so that they can be easily collected. 4.) As the capacity of Synthetic Biology and iGEM is hinged upon the support from the larger public community, the Illinois iGEM team is prepared to facilitate educational sessions, surveys, and ethics panel’s, to better equip the public community to make informed decisions on topics in Synthetic Biology.

Bacterial Decoder and the Registry of Standard Parts

Last year, the Illinois iGEM team worked to construct a decoder function in E. coli using genetic logic gates comprised of transcription factors and sRNAs. A decoder is a low-level computer architecture that produces a specific output or response depending on the combinations of 2n inputs. The team constructed some parts to this design, but failed to complete the final decoder construct in time for the 2009 iGEM Jamboree. Continuing members worked to construct the remaining parts for this decoder and collected data to verify the integrity of these parts. This year’s team intends to 1.) compile the parts to the decoder, 2.) optimize the system by integrating additional parts from the Standard Registry of Parts, and 3.) implement this function into an environmental application.

Synthetic Gene Networks in Synthetic Biology

The engineering of biological systems that process information, materials, and energy holds great promise for developing solutions to many global challenges. The construction of a standard genetic regulator that demonstrates strong bi-stability and facile manipulation poses a significant hurdle to synthetic biologists. The iGEM team intends to look into the construction of artificial ribonucleic protein (RNP) complexes using bacterial sRNA as a model. These regulators will/may be used to construct the necessary biological logic gates in the decoder schematic.

Cell Surface Engineering

Development of a standard part for the expression of proteins on a cell's surface will provide other scientists and engineers with a useful tool to pursue their own research and goals. One of the primary ways in which cells interact with their environment is through expression of proteins and carbohydrates on their outer membrane. The UIUC iGEM Team plans on utilizing this to allow a cell to capture free-floating toxic metal ions in solution. The cell will then enter stationary phase, where a system introduced by our team will cause the cells to float to the surface of the solution for easy collection.

Bioethics and Human Practices in Synthetic Biology

As the Synthetic Biology community grows, major issues including the ethics of genetic engineering, the creation and manipulation of “life”, will need to be addressed. While the horizon of Synthetic Biology promises answers to a myriad of global questions and problems, it does bring with it the possibility of great destruction and terror. The iGEM team will 1.) determine the current understanding of Synthetic Biology of different population pools through the distribution of surveys, 2.) facilitate educational sessions for children, students, and professionals alike, while 3.) engaging in academic seminars with other Synthetic Biologists, iGEM teams, and leaders, to evaluate a proper course for future direction in policy, regulation, and education.

Protocols

Making Electro-Competent Cells

Materials:
• DH5alpha glycerol stock
• Two LB plates
• 5mL LB and 14ml round bottom polystyrene tube
• 500mL LB
• 1L chilled water
• 50mL conical vials
• 40mL of 10% Glycerol
• **Must reserve the table top centrifuge
Directions:
1. Streak out DH5alpha from glycerol stock; grow overnight
2. Re-streak your DH5alpha; grow overnight*
3. Pick colony and grow in 5mL LB overnight
4. Add 2.5mL of the DH5alpha overnight to 500mL of LB
5. Let grow 4-6 hours until the OD is >=0.5
6. Centrifuge 10min. at 8,000rpm at 4degC
7. Pour out supernatant and resuspend in 5mL ice-cold water. Then add 500mL cold water and mix well
8. Centrifuge 10 min. at 8,000rpm at 4degC
9. Pour out supernatant and resuspend in 5mL cold water. Then add 500mL cold water and mix well.
10. Centrifuge 10 min. at 8,000rpm at 4degC
11. Pour out supernatant and resuspend in 5mL cold water. Pour into 50mL Vial and add 40mL 10% glycerol
12. Centrifuge 10min. at 4,200rpm at 4degC.
13. Resuspend in 1mL 10% cold glycerol and mix.
14. Pour into micro-centrifuge tubes and store in -80degC
*This will remove all the glycerol from the cells.

Site Directed Mutagenesis

Procedure modified from [http://www.genomics.agilent.com/files/Manual/200555B_01.pdf Stratagene QuikChange II Protocol]

Materials
PFU Ultra Polymerase (high fidelity)
10X Reaction Buffer
DpnI (20U/μL)
dNTPs
Competent Cells
General Overview
1. Design primers
2. Mutant strand synthesis (PCR)
3. DpnI digestion of template
4. Transform and plate
Primer Design Considerations
• Both primers must contain desired mutation and anneal to same sequence on opposite strands of the plasmid.
• Keep the Primers aruond 25-45 bp long with 10-15 bp on either side of the mutated base.
• Keep the melting temperature greater than or equal to 78°C
Tm=81.5+,41(%GC)-675/N-%mismatch where N is the length of the primer
• Try to keep the primers to a minimum GC content of 40% and end the primer in GC for a clamp.
• Make sure primers are in excess to template PCR

Ingredient	Amount
10X Buffer	5 μL
Template	20 ng
Primer 1	125 ng
Primer 2	125 ng
10 mM dNTPs	1 μL
Polymerase	1 μL
H2O	Bring to 50 μL

Program:

95°C	3 min
95°C	30 sec
annealing temp	1 min
72°C	1 min/Kb
go to step 2	12X
72°C	1 min
hold at 4°C

PCR Purification via Promega Kit
DpnI Digestion

DNA	500 ng
Buffer 4	5 μL
BSA	.5 μL
DpnI	1 μL
H2O	Bring to 50 μL

Incubate at 37°C for 1 hour, inactivate at 80°C for 20 min. Transform via electroporation

Results

Experiment still in progress.