Team:Lambert GA

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• Results/Conclusions
• Safety
• Attributions
• Human Practices
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We are from Lambert High School in Forsyth County, Georgia, just outside Atlanta. We are honored to be Georgia's first high school iGEM team and we are very excited to be part of this group. Our instructor is Mrs. Janet Standeven, who teaches biology and environmental science at Lambert. We are being sponsored and aided in many ways through the extraordinary help of Dr. Mark Styczynski from Georgia Tech's Chemical Engineering department. The members of our iGEM team are very enthusiastic about biotechnology and molecular engineering and are glad to be presented with the opportunity to gain valuable experience in this ever-expanding bio technical field and to have been able to work in doctorate labs at the prestigious Georgia Institute of Technology being just high school students.
	Team Lambert_GA

Official Team Profile

Contents 1 Project 2 Future Aspirations 3 Notebook 4 Results/Conclusions 5 Safety 6 Attributions 7 Human Practices 8 Activities

Project

In 2010, Georgia Institute of Technology’s iGem team submitted the part K410000, a periplasmic heat generator made with HybB and OmpA and AOX. The following year several teams used HybB in their projects with mixed results. Our project is the continuation these projects and the characterization of HybB. To do this we will put RFP under HybB promotion. In addition to we will put OmpA AOX under three constituitive promoters. The purpose is to determine whether the strength of constituitive promoters will change the amount of heat released by generator OmpA AOX. With HybB we are hoping to get reliable results from cold shock treatment.

The purpose of our project is to Biobrick HybB, which is a cold shock promoter. A Biobrick is a sequence of DNA that has standard prefixes and suffixes. The HybB promoter showed up in the Parts Registry and iGEM literature in 2005. The use of HybB has shown mixed results. Our goal is to have a reliable cold shock promoter in order to use temperature to regulate protein expression. We decided to use HybB as a promoter because, though it has only yielded mixed results, it showed promise.

HybB was synthesized for the first time by the 2005 UCSF iGEM team. The 2006 MIT iGEM team standardized HybB and submitted it to the parts registry. HybB has the part number J45503 and is not available from the Parts Registry. Georgia Tech’s 2010 iGEM team engineered a chassis to include HybB, OmpA, and AOX. We obtained HybB from Georgia Tech's 2010 iGEM Team, but upon sequencing and experimentation, we learned that the stock was not reliable.

The 2011 Leuven iGEM team reported, “We see that promoter activity is induced when cells are transferred to 25°C and even when they are put in an ice bath (4°C). Unfortunately, however, cells that are kept at 37°C also display an increase in promoter activity, indicating leakiness in the system.” The 2011 Groningen team reported that the HybB Promoter did not work. Through sequencing, they proved that HybB was found in the cells, and they ran the reactions at the same temperatures. However, they could not get the same results as the Leuven team. Due to these more recent findings, we are not sure whether HybB works or not.

Future Aspirations

By engineering a reliable coldshock promoter, we can provide a switch to turn on gene expression in order to control cell protein expression using temperature.

Notebook

Date	Accomplishments
January 1-15	Met for the first time during class time as an Instructional Focus. Discussed project ideas and iGEM in general. Gave out protocols to study
January 16-31	Practiced transformations with p-GLO from Biorad. Learned about Bacterial Antibiotic resistance using Biorad Antibiotic Resistance Lab.
February 1-14	Discussed project ideas. Planned for visit to Georgia Institute of Technology, Styczynski Lab. Sold Pretzels for fund raiser.
February 15-28	Studied and took test over iGEM protocols and 3A Assembly. Finalized list of Biobrick parts of interest. Outlined initial ideas. Use K410000 to characterize cold shock heat generator.
March 1-14	Field trip to GA Tech on March 9 • Rehydrated J23119, J13002, J04450, K410000 • Miniprepped parts • Ran diagnostic gels • Sent parts for sequencing • Calculated concentration of DNA • Met with team members from GATech iGEM. Discussed the K410000 project. • New idea for project; isolate HybB and make it a Biobrick. GATech team will send their frozen stocks.
March 15-28	Worked on research for the new parts. Wrote Project proposal.
April 1-24	April 1st • Transformed again • Made Amp plates • Cataloged Gaucher 2010 box • Plated the HypB with RFP on Amp plates April 4th • Liquid culture results were analyzed • Took the HypB samples and mixed with Glycerol (Glyceroled Bio Bricks) and placed them in 80oC freezer • Streaked LB plates with HypB samples, promoter, and K410000 (bio brick) • Made more LB plates • Miniprepped hypB parts and K410 from Gaucher box • Calculated the DNA concentration • Calculated concentrations for sequencing • Wrote instructions for Cold Shock April 24th • Nanodropped with DNA concentrations • Ligation ( Protocol) • Digest of RFP, hypB, psBIC3, 410
May 1- May 14	• Transformation • RFP control and 410 RFP grew but only RFP has an expression • Ran transformation efficiency test with Protocol • Prep for experiment- plated 10 plates and inoculated 16 tubes • Experiment for temperature variation was successful but did not prove hypothesis
May 30th	Several of our members attended a safety training meeting, and are now certified in lab safety courses.

Results/Conclusions

What did you achieve over the course of your semester?

Safety

Everybody on the team participated in Lab Safety Training at Lambert High School and took a safety quiz. Additionally, 5 members of the team completed Right to Know, General Lab Safety, Biohazard training and Recombinant DNA Safety Training offered by Georgia Institute of Technology.

As with any project involving culturing bacteria, appropriate safety measure were taken with our project. Though none of the bacteria we work with are human pathogens or environmental liabilities, it is possible that unwanted contaminants could be unintentionally cultured in large volumes. Therefore, we treat every culture as if it were dangerous and take important precautions, such as wearing Personal Protective Equipment (PPE), cleaning up any spills with 10% bleach or 70% ethanol, and autoclaving all culture tubes and plates before disposing of their contents.

Whenever the team was performing procedures, everybody wore safety goggles and gloves. We followed all procedures very carefully, and we were circumspect with cleanup after experiments.

The team was supervised by Mrs. Standeven, Dr. Styczynski, and Mrs. Cochran the entire time we were in the Georgia Tech lab.

Attributions

Who worked on what?

Everyone in the Lambert iGem team is motivated and dedicated to enhance the team and our projects. Mrs.Standeven, has dedicated many hours of her day to help prepare for upcoming labs.

Human Practices

In our inaugural year we focused on educating our teachers, fellow classmates and administration about the purpose of iGEM. We recruited members from the 9-12 grade which gave a large audience with whom to spread the information. Synthetic Biology as a topic was a new idea even to some of our teachers. Several members of our team volunteer at both Science Olympiad camps and STEM camps. They brought activities we had performed as team to the younger audiences in order to familiarize them with DNA.

Activities

Our favorite team snack is Pretzels. King of Pops was a special treat in Atlanta on a work day.