Stanford/BIOE44:Module 4:Day1

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

READ ME (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.

We'll break into two teams for the next four sessions:

• Team Moss will work on bringing a new organism (a plant!) into the BIOE44 lab course. We've never worked with a plant before and so we all have a lot to learn and prepare. Imagine what students will be doing in 5 and 10 years starting from what we build together now.
• Team Arsenic will work to refine and attempt to complete and test construction of at least one arsenic sensor. The arsenic system we starting the quarter with is an exemplar of an engineered genetic environmental monitoring system, and so if we can become expert is working with and characterizing this system, we will all learn many useful things that will help us test your designer DNA encoded systems when they return.

FIRST THINGS FIRST

1. In order to prepare for helping other people to use our parts, please register for an account on the Registry of Standard Biological Parts (click here). Once you have an account email Drew for an access code to our course so that you can connect to the BIOE44 account. You'll be creating an official entry for your designer DNA parts on the Registry later (so let's get the logistics taken care of now!).
2. Go here and decide the top 2 articles you would enjoy reading and presenting on. Please give this information to Drew before 2p today.

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

We have three primary goals:

1. Review and improve the design of the arsencic sensing system, revising the design if needed based on the availability of DNA parts; we'll want to make a great description of the system, at the parts, device, and system levels
2. Complete assembly of at least on integrated DNA construct encoding all the parts of our system.
3. Test and analyze the performance of the system (does it work or not?)

Using a Standard Visual 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 (~140 bp)	cPCR, PCR Cleanup, Digested@EcoR:SpeI, Gel Extract	6ul	~200ng/ul	Koshlan
RBS + arsR ORF (~500 bp)	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) (~600 bp)	cPCR, PCR Cleanup, Digested@EcoR:SpeI, PCR Cleanup	20ul	~40ng/ul questionable quality since no gel extract	Koshlan

Label	Meaning	Concentration
1kb ladder	NEB 1kb ladder
003 with IP	Part K274003 (green generator) cut at XbaI/PstI and gel extracted using Qiagen kit with isopropanol step included.	50 ng/ul
003 without IP	Part K274003 (green generator) cut at XbaI/PstI and gel extracted using Qiagen kit with no isopropanol step.	50 ng/ul
003 Zymo	Part K274003 (green generator) cut at XbaI/PstI and gel extracted using Zymo kit.	~75 ng/ul
100 XbaI/PstI	Part K274100 (red generator) cut at XbaI/PstI, gel extracted with Zymo kit	250 ng/ul
200 XbaI/PstI	Part K274200 (orange generator) cut at XbaI/PstI, gel extracted with Zymo kit	250 ng/ul
pSB2K3 EcoRI/PstI	Vector [pSB2K3] cut at EcoRI/PstI, gel extracted with Zymo kit	150 ng/ul

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