Stanford/BIOE44:Module 4:Day1

M4: Day 1 - "Baking is a Busy Time"

Introduction (ALL BIOE44ers)

OK. You did a great job designing your DNA and getting your synthetic gene synthesis orders placed at DNA2.0, Inc. We expect that your designer DNA will return in time for lab on 20 May. The exacting timing is out of our control, and some parts may be impossible to make; we'll find out!

If the DNA arrives as we expect then we will only have 3 lab sessions to assemble and test you parts. Practically, this means that we all need to work together over the next 4 lab session to prepare for the arrival of your designer DNA, so that we can get right to work on testing it before the quarter ends. Note that this situation is very much akin to working in a "real" research lab.

We have three goals in working together over the next four sessions. First, have fun and learn a lot. Second, be safe. Third, prepare for testing your specific systems. Fourth, leave behind tools and engineering infrastructure that will help the students and others coming later, who will build on your work. Remember that a great engineer doesn't just solve one problem, but helps all other engineers solve their problems too.

Team Moss

Moss

We will learn how to work with Phycomitrella patens. We have 3 main goals:

1. Learn how to culture the moss.
2. Figure out how to transform it.
3. Make freezer stocks for all the future BIOE44 students.

None of us has ever worked with moss before so we will all be figuring it out together. Here are some resources we can start from:

Moss Protocols

• Culturing Moss
• Making protoplasts
• Transformation

References

1. Hohe A, Egener T, Lucht JM, Holtorf H, Reinhard C, Schween G, and Reski R. An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens. Curr Genet. 2004 Jan;44(6):339-47. DOI:10.1007/s00294-003-0458-4 |
2. Horstmann V, Huether CM, Jost W, Reski R, and Decker EL. Quantitative promoter analysis in Physcomitrella patens: a set of plant vectors activating gene expression within three orders of magnitude. BMC Biotechnol. 2004 Jul 7;4:13. DOI:10.1186/1472-6750-4-13 |

Team Arsenic

Using the Same Language

Please review common symbols for representing your device. http://openwetware.org/wiki/Endy:Notebook/Synthetic_Biology_Open_Language

Current Inventory of Parts

Please enter what you have and indicate the state which it is in.

Part	status	volume	notes	contributor
O/P for ars operon	cPCR, PCR Cleanup, Digested@EcoR:SpeI, Gel Extract	6ul	~200ng/ul	Koshlan
RBS + arsR ORF	cPCR, PCR Cleanup, Digested@XbaI:PstI, Gel Extracted	6ul	~80mg/ul (extra codon flanking ATG)	Koshlan
O/P + RBS + arsR (Combined like the edinburgh part)	cPCR, PCR Cleanup, Digested@EcoR:SpeI, PCR Cleanup	20ul	~40ng/ul questionable quality since no gel extract	Koshlan

Refining our Goals

We need to think about the fundamental relationship between the amount of pollution in our sample and the reporter strength. What will this relationship look like? How can we alter it? Consider the effect of the following.

1. copy number
2. ribosome binding site
3. ratio of ArsR transcription factor to the operator site
4. one plasmid or multiple plasmids
5. what else?

What are our options.

Here are some imaginary curves to stimulate your thinking.

Deconstructing our Device

What do we need? Should we shuffle anything?

Design and Baking