Pushing the P1

Introduction

Having a lab space and instruments is one thing, possessing the tools to do real biology is another thing. For close to two years now, the Hackuarium community has built a space, where people can work on projects related to biology among other fields. The pretension of the international DIY bio scene, or closely related biohacking scene, is to bring to the people the tools to perform and work on the whole range of biological applications, as qualitatively and cheaper than what the industrial or academic institutions do. To achieve such level, the Hackuarium lab has to be upgraded to a higher level of competency. This level is the P1 biosafety level where genetic manipulation open the door to a vast range of research topic and engineering opportunities. This scientifically enriching, yet delicate step, is now in motion but in order to start practicing several items involving legal, technical and community topics have to be settled. Indeed, our laboratory practices and transparency as a citizen lab have to be absolutely irreproachable. The challenges we face are described in this document.

Timeline

Here is the basic timeline we will try to keep in order to make the project move on. It is obviously a "best case scenario" approximation.

Proposed timeline for the project

Pivot Points

Here we develop and tackle down the most critical points of this project. The idea is to estimate if our space can accommodate such an infrastructure and if our community has the shoulders to carry the necessary responsibilities. The points have to be assessed keeping in mind the first standardized lab procedure that will be globally used by the lab (see generalized procedure).

Lab room and material

Here we discuss the size features and needed characteristics of the room we are going to use for the lab. We also describe a material list we would need to equip the lab to the minimum.

Material

Stockage:

• Congelateur -80°C, -20°C
• Frigo, 4°C
• Glace

Transformation:

• Heatblock statique
• Heatblock shaker

Culture:

• Incubateur statique
• Incubateur rotatif (pouvant refroidir)

Post-culture:

• Centrifuge large volume
• Ultra centrifuge
• Sonication
• Centrifuge 1.5ml
• Autoclave destruction

Mobilier:

• Benches
• Armoires

Autres:

• Bec Bunzen
• Sturdy trash box
• Safe liquid waste flask

Waste eliminations

Here we describe who can and accepts to eliminate our wastes at what cost and try to estimate the frequency of elimination. We base the frequency on a per project base. How many liters of cultures are produced per production? How many biowaste bag can be filled in a week? etc...

Calculated base costs

Here we estimate the cost of starting the whole setup. Taking a standardized transfection method, expression method, cell line and antibiotic for the whole lab, how much would it cost to start it up? What are the most important enzymes? Are there opensource alternatives?

Softwares and Databases

Here we discuss the gene editing softwares to design plasmids, primers and gene building blocks. A total transparency has to be implemented in order for people to follow our activities. What kind of database will we use to store the ordered primers sequences? Same for the plasmids?

Providers

Here we discuss the possible service providers, their costs and if they are ready to deal with us. We canot create plasmids without primers or genes and therefore need providers. We cannot confirm the authenticity of our work without a verified plasmid sequence and therefore need sequencing services.

Legal

Hackpad first exploration document : https://hackpad.com/Wanna-go-P1--I8F1GOCYTR9
As said previously our laboratory practices have to be irreproachable. We carry the "hacker" label and therefore, for the public opinion and the built of trust, cannot fuck around. We need to study in depth several aspects of the legislation governing genetic manipulation.

Generalized procedure

These procedure will be implemented in the lab as first benchmark in order to have a work basis. They are optimized for cost & material efficiency.

Bacterial

Strain: e.coli Plasmid: Resistance: Ampicilin

Protein expression

• e.coli chemo competent cells will be stored in a -80°C fridge.
• Transformation will be made by heat shock as described in ("doc name here").
• Classical LB-Amp plating with static overnight 37°C incubation.
• Liquid cultures can be either made as:

• LB-Amp followed by IPTG induction.

(-) IPTG is expensive

(-) Monitoring of optical density requires time + UV-spectro

(-) Large volumes (1L medium in 5L flasks) for decent yields + large capacity incubator cooling to 16°C = more waste

(-) Have to centrifuge in 500ml flasks = more post-culture work.

(+) yields are often good, procedure is bulletproof

• Modified auto-inducible Terrific-Broth (TB-of-doom)

(-) more complex medium preparation (can still be made as large stock)

(+/-) yields are good but proteins might be hard to extract

(+) No UV monitoring ("by eye check"), after growth expression can be made at room temp.

(+) small 100ml cultures in 500ml flasks

(+) all post-culture fork can be made in 2x50 ml flasks (convenient)

• Purification can be made by single His tag purification

• We have more than 200ml of Ni-NTA resin
• The resin can be recycled easily
• Quite simple procedure (requires 3 buffers)

(-) Single His-tag purif will not yield absolutely pure proteins

• Concentration

problem remains to be solved

• Storage will be done at -20°C in 40% glycerol