HTGAA 2021: Bio Production (DIY Gingko)

Guest Lecturer : Patrick Boyle (Ginkgo Bioworks)

TA : Pat Pataranutaporn (MIT), Sebastian Kamau (MIT), Eyal Perry (MIT), Alex Mijalis (Harvard)

Background

Lycopene is the red pigment that gives tomatoes their color. This pigment is also made by microbes. In fact, transferring a 3-enzyme pathway to E. coli can convert farnesyl diphosphate (FPP) to lycopene. The computational tools and databases presented today can also be used to enhance lycopene production in E. coli or even produce different colors, such as the beta-carotene pigment that makes carrots orange.

For the laboratory portion of this assignment, you will characterize lycopene production in the E. coli strain from Addgene with the pAC-LYC plasmid. You will also characterize beta-carotene production in E. coli from Addgene with the pAC-BETAipi plasmid. Note, the pAC-LYC plasmid contains three genes from Erwinia herbicola: CrtE, CrtI, and CrtB. The pAC-BETAipi plasmid produces beta-carotene through the addition of the Erwinia herbicola crtY gene. All plasmids include the gene for chloramphenicol resistance.

Lycopene and other Carotenoid Production Pathway

Plasmids involved in the experiment

Assignments

Recitation Slides


Design and run an experiment to understand how different factors influence bioproduction.

The available factors are genes (pAC-LYC, pAC-BETA), media (LB, 2YT), concentration of precursor (fructose), temperature, and shaking. Please follow the steps below to execute the homework:

Template

The template for experimental design & code for running the experiment on the robot is available here (please make a copy of the file to your own google colab) : https://colab.research.google.com/drive/1pDFV0ADd6JsdiVtcbnKFKFQVVU0NjLVb#scrollTo=sBrB_Z0rVxIg

Scheduling

The student can book the slot to run the experiment on the robot via : https://docs.google.com/document/d/1QwQ3-uxkhSwQnZfEciwykrY10r0up1ikrWt2KNn7btk/edit?usp=sharing

The available factors for the experimental design are genes (pAC-LYC, pAC-BETA), media (LB, 2YT), concentration of precursor (fructose), temperature, and shaking
Experimental Setup
DIY Shaker

Identify a cool gene (related to your final project) and try to order it via twist bioscience following the video instruction bellow. Please select "Clonal Genes" for the gene type. Once you finished uploading your gene sequence, select a vector (please refer to Sebastian's slide for more information), and perform codon optimization (if necessarily), please download the GenBank file of your gene (+ vector) and send it to here : https://drive.google.com/drive/folders/1yX2VgEaWOb29Kx-B_jswn39Zx-m8N3Vx?usp=sharing
Download your sequence as a Genbank file

Twist Bioscience Video Instruction

Write a short paragraph responding to "If you have the capability of Gingko Bioworks foundry, what would you do and why?" These could be COVID19 related projects or broader synthetic biology projects. Ginkgo Bioworks is taking proposals to leverage the use of their platform to support technical projects; how might you leverage Ginkgo’s technical platform to support your project? Write a description of how you would utilize their platform. What tools and capacities would you use? How would you use them? How would the use of Ginkgo’s platform accelerate or increase the technical capabilities of your project?

Related Readings & References

Blogposts and other resources

Other Useful Resources