Synthetic biology project invite

[Avery louie]

Hello Everyone,

In the past, there has been interest in a more advanced "synthetic" or molecular bio class involving not only adding foreign dna to new chassis, but actually manipulating the DNA, as far as restriction digests, ligation, and PCR.  I am working on developing this course, but just like the last class that was developed, I want to invite a few people to join me on this project.

What:

We will start by growing different bacteria that have genes that we want.  Then we will clone the genes out using PCR, and eventually digest and ligate them into a plasmid.  We will take that plasmid and put it in e. Coli.  BAM!  We will have made a transgenic organism with our own cloned DNA.  I will be cloning a pigment gene from a Rhodococcus, and you are welcome to do that as well, but it would be fun to do a few genes- maybe a lacZ' or an ecoR1.  I have done this before, but not at bosslab.

Who:

Five people with a little bit of molecular biology background (biotech 101, read a book, experience reccomended but not required), who have $150 to spend on this project, and lots of time weekday nights.  Money goes to reagents for PCR, primers, ligase, pcr cleanup, and twoards a scale/uv vis spec for dna quantification.

When:

When you can come in, but realistically we can break tasks up so that people can skip days and have their lab mates cover them.  Once the parts are in, you can even complete the project on your own time, if you feel comfortable.  The project will start when enough people to do it have been collected.

If you are interested, just email me or reply all to the list.  Project will begin when there are enough people.

--Avery

[Margret Storm]

Is there a physical location, or can people from anywhere join in?

[Avery louie]

I am not sure I understand you.  We will be transferring a gene from a wild type rhodococcus to a plasmid, and the plasmid into e. coli.  Kind of like a biobrick part, minus the standard ends.  LacZ' fragments are also genes of interest for use in constructing plasmids- it has a pretty easy functionality to test for, so it might be fun to yank out of e. coli and stick into a plasmid.  Or a restriction enzyme producing plasmid- that could be fun.

You are completely correct about the pigment vs. lacZ+X-Gal argument though.  It would make a sweet cloning vector.

There is no need to score a "pigment + phenotype" because we are copying genes out of rhodococcus into e. coli, and the rhodo should have the gene (it has been sequenced).

--A

On Mon, Jun 4, 2012 at 4:44 AM, Srdjan Gavrilovic <sasvim...@gmail.com> wrote:

Hi Avery,
Im sorry but I cant join you organizing the class but I have few suggestions to make.

"a pigment gene from a Rhodococcus"..."I have done this before, but not at bosslab."

Im not sure how easy it is to distinguish transgenic bacteria from wild type. If scoring is easy I would say that it would be a great idea to try and engineer the gere for future cloning eforts.
The thing is that for cloning into LacZ, people have to use blue-white screening. To score a potentially right clone use of IPTG and X-gal is needed. Getting and dissolving it is a real pain.

Adding multi cloning site into a pigment gene and redesigning any of cloning plasmids (exchanging LacZ with MCS for a pigment gene with MCS) would be a great brake through for DIYbio. People could start doing cloning easier.
Restrict plasmid, attempt to ligate an insert, transform into e.g. E.coli and take colonies that do not produce pigment (if pigment is present it means that ORF is restored and there is no insert), test if they have the right insert and do what ever they want to do with their gene of interest.

I do think that its nice to educate people ubut showing them how to clone plus building a great tool for the DIYbio people at the same time. I would say that you might get a lot of people wanting to help and to have such a plasmid for private cloning purpose. Taking a course and going home with a plasmid like that...Even academic labs would want to have such plasmid.

Now the thing is. How easy it is to score a pigment positive phenotype in E.coli?

[Avery louie]

The location is BOSSLAB, in Boston, but if you want to get involved, the protocols will be online, and the plasmid will be released when it is done.  It would be fun to do some kind of parallel experimentation if you have a lab as well.

--A

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[Srdjan Gavrilovic]

I agree for restriction enzymes. It would be great to have them just be sure to have corresponding modification partner. Without correct methylase many restriction nuclease will cut host DNA and kill them before you can do anything useful.
It would also be grate to have other enzymes (e.g. polymerase, polynucleotide kinase, phosphatase, DNA ligase. All of them required for serious cloning project).

I was thinking about scoring presence of an active "pigment gene" in the E.coli vs a gene with interrupted ORF. I didnt look in details about this gene and I wanted just to see if you have an idea how efficient it would be in E.coli for the purpose of cloning at first place.
How strong is signal/pigment/color in E. coli?

LacZ' is out there for ages. It is easy to get it but we can work together to make new and better things. You can demonstrate principles and in addition make a great tool.

If you can give me accession no. of your gene and sequence of the plasmid you are planing to use I can look into that. Im from Europe and I cant get involved physical but I think that I still can contribute.

Cheers, Srdjan

[Alex Hoekstra]

I'm game, of course, but you did sort of skip the "Where" aspect of the group project.  Having online protocols so that folk from around the world can follow along on their own is nice, and of course some contributions can be made through decentralized chatting, but a lot of this work calls for in-person collaboration.

On Sunday, June 3, 2012 4:53:40 PM UTC-4, Avery wrote:

[Srdjan Gavrilovic]

On Monday, June 4, 2012 5:02:29 PM UTC+2, Alex Hoekstra wrote:

I'm game, of course, but you did sort of skip the "Where" aspect of the group project.  Having online protocols so that folk from around the world can follow along on their own is nice, and of course some contributions can be made through decentralized chatting, but a lot of this work calls for in-person collaboration.

No, I did not miss "Where" part.
My point was to coordinate and try something new together. Reinventing wheel will not take us far.

Cloning might be done one day and colonies could be screened next day. After that plasmid can be isolated and blotted onto a peace of paper and sent to someone else to improve it (eg to add MCS) and there you have it a plasmid that rocks and can be distributed all through the community.
For this to work we have to find a way to do it in such manner that a product of one step can be used for the next.

I know that I'm not able to reach there for lab work but I might be the one to take it up after and I just want to see if we can organise it or should we do things everyone for our self and just keep talking about organisation.

[Avery louie]

More details about plans for putting it in a vector and sequences will be avalible soon, but mostly I am looking for people who want to collaborate at bosslab.  Two reasons:

1.  make cool stuff for fun.  It is easy to PCR genes out of plasmids, given that you have the right primers.  Primers can even be used to attach new sticky ends to a sequence- lets say you have hindIII-RBS-pigment-stop-EcoR1, you can make SacI-RBS and stop-XhoI primers and do as you please with the DNA.

2.  equipment shakedown @ bosslab

3.  show people how to steal genes from the environment.  Your floor is littered with thousands and thousands of bacteria, and each bacteria is full of DNA.  Surely there must be something interesting down there...

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[Mega]

That's an awsome idea....

Because as a DIYer it's very difficult to get X-Gal. I either as my proffessor (again and again and again, he'll hate me :D ) or buy it on the internet. Some companies won't ship it to residental adresses, and if they do, delivery costgs are enormous. And also the X-Gal won't be cheap.... A pigment, on the other hand is synthesied out of nothing than LB-Medium. That would be a cheap, most-easy-to handle way of reporter gene!!


I think it is possible to design primers for PCR to cut the gene into two genes. Handle if as the gene were two genes. So it will be cut in the middle, won't it. There the primer should have the sequence of a restriction site.

But maybe there's another problem: If the vector plasmid has MCS, there may be 2 of each restr-site?? (If you take the RE in the middle).