Wanted Bacteria: Dead or Dead The development of a kill switch that - - PowerPoint PPT Presentation

Wanted Bacteria: Dead or Dead

The development of a kill switch that activates upon a cell escaping from the laboratory setting into the surrounding environment

The Public Fear of Genetically Modified Organisms ¡

Development of a Kill Switch ¡

The Trigger ¡ The Outcome ¡ ¡ Exposure to UV Radiation Expression of the ccdB Gene

Two Competing Designs ¡

¡ for a Kill Switch

¡ ¡ ¡ ¡ ¡ ¡ ¡ ¡The “Simple” System vs. The Riboregulator System ¡

The more straightforward approach The more complicated “lock and key” approach

The Simple Kill Switch ¡

The Results ¡

The ¡Simple ¡Kill ¡Switch ¡

UV ¡Promoter ¡ recA ¡Promoter ¡

recA + ccdB ¡

▪ The ccdB translational unit was synthesized as a string. ¡ ▪ Attempts to insert the part into a backbone ultimately proved tricky. ¡ ▪ Subsequent attempts at ligations with the recA promoter via 3A assembly also proved diificult. ¡ ▪ Ultimately, the simple construct failed and no construct with ccdB was produced. ¡

recA + GFP ¡

The Cis-Repressing and Trans-Activating Riboregulator System ¡

Taken from “Engineered riboregulators enable post- transcriptional control of gene expression” by Isaacs et al.

Riboregulated Kill Switch ¡

Cis-Repressed Construct ¡ Trans-Activating Constructs ¡

Riboregulated Kill Switch Flow Chart ¡

The Results ¡ ¡Riboregulator System

▪ We ¡were ¡unable ¡to ¡transform ¡the ¡synthesized ¡plasmid ¡from ¡Life ¡

• Technologies. ¡ ¡

▪ It ¡took ¡3 ¡to ¡4 ¡attempts ¡to ¡merely ¡synthesize ¡the ¡plasmid. ¡ ▪ We ¡purchased ¡NEB ¡cells ¡that ¡were ¡reported ¡to ¡be ¡ccdB-­‑resistant. ¡ ▪ As ¡a ¡result, ¡our ¡mentor ¡contacted ¡Tom ¡Knight ¡for ¡his ¡personal ¡ccdB-­‑ resistant ¡cells, ¡which ¡we ¡used. ¡ ¡ ¡

The Results ¡

Riboregulator System

We successfully cloned recA+ trans + dterm ¡ into a chlor backbone! ¡

But what happens if there’s no UV light? ¡

Alternative: The No-Arabinose Kill Switch ¡

The Trigger ¡ The Outcome ¡ ¡ The Absence of L(+) Arabinose Expression of ccdB Gene

The No-Arabinose Kill Switch ¡

BioBrick Parts Used ¡

• 1. ¡
• 2. ¡
• 3. ¡

Our Constructs: ¡

No-Arabinose Kill Switch Flow Chart ¡

The Results

▪ We attempted the ligations of the AraC construct to the pBad construct. ▪ We also attempted the ligations of pCI to a ccdB translational unit ▪ However because we were running out of time, we decided that characterizing the pBad construct would be a more effective use

• f our time.

The Results ¡

For characterization: ¡ Road Blocks

• Wrong Sugar: Used D-arabinose instead of L-

arabinose

• Wrong concentration?
• Serial dilution of Arabinose
• Wrong cell strain:
• AraD139 mutation prevents cell from

metabolizing arabinose. The entire L- arabinose

• peron is essentially the metabolism of L-
• arabinose. pBad doesn't work unless the L -

arabinose operon works. ¡

Sequence Results of Characterizing Construct ¡

Summary of Project

• 1. Simple Construct

– RecA and GFP was successfully ligated, but unfortunately not in a chlor backbone. – RecA and ccdB was not ligated due to problems with the ccdB gene itself and problems as a string ligating simply into a plasmid backbone.

• 2. Riboregulator System Kill Switch

– RecA and Trans was successfully ligated and submitted as a part. Unfortunately unable to characterize because the other component of the riboregulator system had problems. Synthesize plasmid created for such system had problems ligating to a chlor backbone.

• 3. No Arabinose Kill Switch

– Successfully ligated pCI with pBad construct. Unfortunately we were unable to transform into a cell strain without AraD mutation

Acknowledgements ¡

We would like to thank all the faculty of Stuyvesant High School for the resources and invaluable guidence they provided. Acknowledgement also goes out to Andrei Talaba, Camilla Vincent and David Chen, who helped with lab work, and Kelvin Mei, who helped with coding, who were unfortunately unable to be here today. Thanks to Zara Levanthal for designing our logo. Thank you Cooper Union iGEM team for sharing material when

• ur reagents and other materials started running out!

Special Thanks ¡

A very special thank-you goes out to our mentor Dr. Ellen Jorgensen, the founder of Genspace Biolabs. She provided indispensible advice to the team on how to do much of our lab work and what direction to go with research, as well as advice on safety and encouragement throughout the competition. ¡

So what have we learned from all this? ¡

▪ Procedures in scientific papers that sound foolproof are not that easy to replicate ▪ If you bring food to lab, always expect to lose 40% to your mentors and labmates. ▪ Your parents will let you stay late in the lab if you’re good at explaining your project to them. ▪ There will be roadblocks and potholes in the process of your project. But keep persevering, don't have a nervous breakdown, and keep calm and carry on.

iGEM is more important than sleep. ¡

But naps are okay! Katty ¡Wu, ¡President ¡ Jacky ¡Cheung, ¡Vice ¡President ¡

Team Members ¡

Katty Wu, President Jacky Cheung, Vice President Qiu Chang Wu, Animations and Graphics Jorge Reyes Shi Hui Fiona Ng Miranda Hallea Justin Chen ¡

A Tremendous Thank You to Our Sponsors!