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Hypothetical Synthetic Plasmid

Microbiolgy Plasmids

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#1 Koeng

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Posted 02 July 2012 - 04:14 PM

Hello! I am bored this summer, so I decided to make a hypothetical synthetic plasmid sequence. I wanted this plasmid to be as SMALL as possible. It took quite a while but I completed a possible plasmid. This plasmid is based off the pUC19 vector. I took out the lacZ gene since I saw it as unnecessary, even though it might turn the bacteria blue. Another reason I took out the lacZ gene is because I want just ampicillin in the agar plates for transformation because it is cheaper Posted Image. I also saw that there was quite a bit of space in-between the genes, and since a lot of this is useless, I took it out. I still wanted some spots for restrictive enzyme digestion, so I put those in-between the origin of replication and the "bla" gene (for ampicillin resistance). I wanted those enzymes to be easy to get, so I choose Hindlll, BamHI, and EcoR1, which are in the DNA in about the middle, the sequence fragment is gaattcggatccaagctt. If there are any nice, short Multiple Cloning sites from any other plasmids that I could add in would be greatly appreciated. I am no expert on protomers, but I believe the ones in the original sequence would work so I didn't change their spots. Any input would be great!

The ampicillin resistance gene (bla) is 641 - 1501and the origin of replication is 1 - 553. The "bla" protomer is 572 - 576 (TTCAAA) and the other "bla" protomer is 592 - 597 (GAGACA). I set it up on ApE (a plasmid Editor) but I can't upload that type of file, nor can I upload a map Posted Image. But I did put the sequence in a nice text edit file. Thank you in advance!!!

-Koeng

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#2 pito

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Posted 03 July 2012 - 01:22 PM

Not sure what you expect from us.

But do you know why lacz is important?
Also: what do you mean with "because I want just ampicillin in the agar plates for transformation because it is cheaper "

It would be better if you could upload a picture of the plasmid showing all the important genes/MCS etc? cant you make a nice figure with the program you used?
If not, there are other, simple, programs out there that let you "draw" your own plasmid: its pretty straight forward, you just enter the genes/MCS that you want and the program draws the plasmid for you.
(its nothing more then a circle with the names of the genes on it; but it might be helpfull since it gives you a visual idea of the plasmid)

PS. whats a ""bla" protomer" ? You mean bla promoter?

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 Koeng

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Posted 03 July 2012 - 03:20 PM

Yes I know what lacz is important for. (Lactose metabolism) I mean when I say "I just want ampicillin in the agar plates" because if I had the lacz gene, I would have to put X-gal in the solution to make it turn blue. And yes I meant bla promoter. I would just like some input, like "you should do this or that", to continue this fun project. (well, fun for me). Maybe some nice MCS sites from different plasmids, if you remember that plasmid just say it and post I can look it up. Or maybe a different resistance, because that resistance gene is better or shorted or because personally when you where in high school the ampiciilin in your plates didn't work but Kanamycin worked really well.

Also thanks for the idea of some new programs, I am looking into them and will probably spend the rest of the night making a really nice map. But right now I'll just paste the pdf of the map I make with ApE.

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#4 phage434

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Posted 03 July 2012 - 04:31 PM

Your plasmid is quite similar to the pared down and repurposed pUC19 derivative I designed for Biobrick work: http://partsregistry...tle=Part:pSB1A3
We added transcriptional terminators surrounding the cloning site, which are very helpful in a high copy plasmid containing a strong promoter. People have gone to a lot of trouble to get rid of the restriction enzyme sites you are putting back in. For cloning vectors, the fewer sites you have (outside of the cloning site) the better. Here in the future, the MCS is also far less useful than it once was. Using a standard set of enzymes in contexts that always work makes much more sense.

#5 pito

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Posted 04 July 2012 - 02:29 AM

Yes I know what lacz is important for. (Lactose metabolism) I mean when I say "I just want ampicillin in the agar plates" because if I had the lacz gene, I would have to put X-gal in the solution to make it turn blue. And yes I meant bla promoter. I would just like some input, like "you should do this or that", to continue this fun project. (well, fun for me). Maybe some nice MCS sites from different plasmids, if you remember that plasmid just say it and post I can look it up. Or maybe a different resistance, because that resistance gene is better or shorted or because personally when you where in high school the ampiciilin in your plates didn't work but Kanamycin worked really well.

Also thanks for the idea of some new programs, I am looking into them and will probably spend the rest of the night making a really nice map. But right now I'll just paste the pdf of the map I make with ApE.

I see what you are trying to do, however: there isnt a lot of "use" when you do it like you do it.
I know that you are trying to improve the plamid, but the problem is that you dont have a "goal", I mean: you can indeed make the plasmid smaller, but whats the point of there is no goal or meaning.

Maybe you should try to look into more practical things like: how can I adjust a certain plasmid to make it a plasmid that I can use for both gram + and gram - bacteria?
Or can I make a plasmid that can be used as a indicator for a certain transformation (eg: how can I show that the insert in indeed present in the plasmid?)
Or like you allready mentioned: what antibiotic gene should be used and why? For example: gram + against gram -: is this the same antibiotic? Or what with antibiotics that are light sensitive...? What about the price of the antibiotics? WHat about the "safety" of the antibiotics...
I know this is a lot of information, but if you have some time and you think its fun, you could try to figure out what "parts" of a plasmid are important and what possibilities are out there.
(eg: what do you need: promoters, markers, MCS, origins.....)


You see what I mean?

Edited by pito, 04 July 2012 - 02:29 AM.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#6 Koeng

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Posted 04 July 2012 - 12:21 PM

Thank you Pito! I do have all summer and this is enjoyable, and I am going to see if I can create a plasmid with a better purpose then just being small. I will post when I complete it. Thank you!!!

-Koeng

#7 pito

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Posted 04 July 2012 - 01:18 PM

Thank you Pito! I do have all summer and this is enjoyable, and I am going to see if I can create a plasmid with a better purpose then just being small. I will post when I complete it. Thank you!!!

-Koeng


Its up to you...
Creating a small plasmid is fine.. but its better to know what the most important elements are and when you need them.
Just think about all the options you have and if you are able to "make" plasmids, thinking about what you need and when you need it, you would be able to do much more then just creating a "small" plasmid.
+ then you would learn/know a lot more about bacteria/microbiology in general because you would need to think about what promoters are, what the difference is between gram+ and gram- , why would you need lacZ too, how to generate shuttle vectors .....

And keep in mind: its not just about 1 plasmid.. its about the right plasmid at the right place.. altough: you could indeed try to create "the ultimate" plasmid that you can use anytime anywhere (without worrying too much about the size)... and then use this plasmid and remove the parts of this plasmid that you wouldnt need for a certain situation...
(its a bit like working back/in reverse: you create the super plasmid that can be used everytime and you just look what pieces you can remove in certain situation)

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#8 pDNA

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Posted 16 July 2012 - 11:52 AM

to be honest ...i did not had a very thorough look on your sequence
BUT i think you wrecked the origin of replication! ...i do no longer see the RNAII -10 and -35 box ...therefore the promoter is gone and i would assume that no replication of that plasmid is possible in E. coli!

So, i would not have this plasmid synthesized ...it would be a waste of money!!!

The origin of replication is the key element of a plasmid ...therefore it has to stay intact! Unfortunatley, most plasmid sequences are not annotated properly (most of the time the annotation just says origin) ...people must keep in mind that the origin of replication encodes two regulatory RNAs (RNAI,RNAII ...in the case of COLE1-like plasmids, e.g. pUC, pET) both having its own promoter. Due to that most people are unaware of the nature of this mysterious region in plasmids.

Small plasmids are important for therapeutic applications (DNA vaccines, gene therapy) ...and removing non-essential regions is essential (best thing is to get even rid of antibiotic resistance gens ...i spent the last 4 years on this issue ...but thats a different story) ...therefore i really like your approach!!!

Best regards,
p

Edited by pDNA, 16 July 2012 - 11:53 AM.


#9 TaylorDNA36

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Posted 13 August 2012 - 10:12 AM

Hi Koeng,

Making new plasmids from scratch is not always an easy task, and there are lots of different elements to consider beforehand. But as some of the other posts have mentioned, figuring out why you are making it is key to making a good vector. If you take a look at biobricks (http://biobricks.org/) or oxford genetics (http://www.oxfordgenetics.co.uk/) you can see how the plasmids are designed to do XY and Z before the vectors were engineered. Check out the drop down box at the latter site and you can see what the guys who made it were trying to incorporate, but they must have sat down and decided this at the start! Good luck with the vector, hope its works out.

Regards,

T

Edited by TaylorDNA36, 13 August 2012 - 10:13 AM.


#10 ascacioc

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Posted 13 August 2012 - 12:26 PM

Interesting summer project. However, I know at least two other companies that did it before you:
pETite vector: http://lucigen.com/s...sion-System.pdf
It is bigger than yours, but I think you cut a bit too much.
I also know another company I used to work in that developed small vectors for its own use. They had cut a bit more than the pETite vectors, but still, in the end they had around 2.3 kb but they had inserted a ccdB gene in the MCS: http://www.ncbi.nlm....pubmed/20646988

@ everybody else whose critizicing the use of such a vector: those vectors are great to work with for transforming large DNA libraries like epPCR for huge genes of >3kb. Usually in directed evolution/protein engineering, this is the limiting step in obtaining a large library.

Since this design is a bit risky, I wouldn't go for ordering the synthetic plasmid, but for 'butchering' the original one. You might find Gibson assembly quite useful in the process:
http://www.neb.com/n...roductE2611.asp (do not buy the kit because it is pure theft; rather buyt the 2 enzymes you need seperately and do it according to the original paper:
http://www.ncbi.nlm....pubmed/19363495
http://www.ncbi.nlm....pubmed/21601685

Andreea





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