Talk:CH391L/S13/DnaAssembly

From OpenWetWare

< Talk:CH391L/S13(Difference between revisions)
Jump to: navigation, search
Current revision (23:14, 14 February 2013) (view source)
 
(15 intermediate revisions not shown.)
Line 4: Line 4:
*'''[[User:Aurko Dasgupta|Aurko Dasgupta]] 20:51, 7 February 2013 (EST)''':I don't have any clear idea of the kind of budgets one should expect for making a 3000bp plasmid in lab, but getting [http://www.wolframalpha.com/input/?i=4+ug+%2F+%28660ng%2Fmol+*+6000%29 1 millimole] of plasmid for ~$1200 actually sounds like a decent deal. Would it be reasonable to say that the labor and materials to make such a plasmid would amount to more than $1200, assuming a few mistakes along the way?  
*'''[[User:Aurko Dasgupta|Aurko Dasgupta]] 20:51, 7 February 2013 (EST)''':I don't have any clear idea of the kind of budgets one should expect for making a 3000bp plasmid in lab, but getting [http://www.wolframalpha.com/input/?i=4+ug+%2F+%28660ng%2Fmol+*+6000%29 1 millimole] of plasmid for ~$1200 actually sounds like a decent deal. Would it be reasonable to say that the labor and materials to make such a plasmid would amount to more than $1200, assuming a few mistakes along the way?  
**'''[[User:Max E. Rubinson|Max E. Rubinson]] 22:47, 7 February 2013 (EST)''':That's probably an issue between you and your boss.
**'''[[User:Max E. Rubinson|Max E. Rubinson]] 22:47, 7 February 2013 (EST)''':That's probably an issue between you and your boss.
-
 
+
**'''[[User:Gabriel Wu|Gabriel Wu]] 00:58, 9 February 2013 (EST)''': From people who do this kind of work, they tell me that assembling a 3 Kb plasmid takes about 3 full days of lab work.  The cost of Gibson master mix from NEB is $154.00 for 10 reactions ($15 for one reaction). The cost of Phusion master mix is $170 for 100 reactions ($8 for 4 PCR reactions). Plates, media, and competent cells add a marginal cost (I would estimate generously no more than $20). Finally, oligoucleotides are about $15 a piece. This assembly would probably take about 8 long oligonucleotides ($160). Then finally, think about how much you make and add your 3 day cost. I estimate generously at about $300 for three days work.  Add it all up, you get about $503. A two-fold difference at I think reasonably generous estimates. It becomes a question if whether your time is beter spent in other places and how quickly do you need the part.
 +
 
'''[[User:Gabriel Wu|Gabriel Wu]] 13:10, 4 February 2013 (EST)''': These are notes to myself. They help illustrate techniques in cloning or trends in DNA synthesis
'''[[User:Gabriel Wu|Gabriel Wu]] 13:10, 4 February 2013 (EST)''': These are notes to myself. They help illustrate techniques in cloning or trends in DNA synthesis
Line 24: Line 25:
*'''[[User:Neil R Gottel|Neil R Gottel]] 13:11, 7 February 2013 (EST)''':I was looking into Ginkgo Bioworks because [http://www.sciencenews.org/view/feature/id/347263/description/Factory_of_Life this article] at sciencenews mentioned they rely heavily on their own special CAD tools when designing their organisms, and found their blog. It hasn't been updated in a year, but their [http://blog.ginkgobioworks.com/2012/01/14/commercial-gene-synthesis/ last post] was talking about how slow and variable the turn-around times are for gene synthesis. They've got a chart showing the length of each order, and the time it took between when the order was placed, and when it was received. Some of their genes (or "synthons" as they call it, since some sequences are not whole genes) took more than two months to synthesize!
*'''[[User:Neil R Gottel|Neil R Gottel]] 13:11, 7 February 2013 (EST)''':I was looking into Ginkgo Bioworks because [http://www.sciencenews.org/view/feature/id/347263/description/Factory_of_Life this article] at sciencenews mentioned they rely heavily on their own special CAD tools when designing their organisms, and found their blog. It hasn't been updated in a year, but their [http://blog.ginkgobioworks.com/2012/01/14/commercial-gene-synthesis/ last post] was talking about how slow and variable the turn-around times are for gene synthesis. They've got a chart showing the length of each order, and the time it took between when the order was placed, and when it was received. Some of their genes (or "synthons" as they call it, since some sequences are not whole genes) took more than two months to synthesize!
 +
 +
*'''[[User:Gabriel Wu|Gabriel Wu]] 00:44, 9 February 2013 (EST)''': So, this is where DNA assembly can fail.  Depending on the number and stability of hairpins, the GC content, and whether or not there are inverted repeats (which leads to homologous recombination) in your sequence, synthesis companies can have a hard time making your DNA order. The advantage to using these services is that you don't have to deal with these things in the lab to construct them. However, synthesis companies have improved their ability to pre-screen these type of difficult sequences and often times will not accept the order at all.  Gingko has actually moved away from ordering full assemblies and instead order oligonucleotides and then assemble the DNA in-house.
'''[[User:Thomas Wall|Thomas Wall]] 20:23, 7 February 2013 (EST)''': I had a formatting issue Gabe, You put CPEC and splice under gibson cloning, making it look like it is a subset of that when I think you mean for it to be a subset of overlap cloning.
'''[[User:Thomas Wall|Thomas Wall]] 20:23, 7 February 2013 (EST)''': I had a formatting issue Gabe, You put CPEC and splice under gibson cloning, making it look like it is a subset of that when I think you mean for it to be a subset of overlap cloning.
 +
 +
*'''[[User:Gabriel Wu|Gabriel Wu]] 01:02, 9 February 2013 (EST)''': Fixed.
'''[[User:Thomas Wall|Thomas Wall]] 20:31, 7 February 2013 (EST)''': This is a cool extension of CPEC I want to potentially start using in my lab (http://www.ncbi.nlm.nih.gov/pubmed/21293463)
'''[[User:Thomas Wall|Thomas Wall]] 20:31, 7 February 2013 (EST)''': This is a cool extension of CPEC I want to potentially start using in my lab (http://www.ncbi.nlm.nih.gov/pubmed/21293463)
'''[[User:Max E. Rubinson|Max E. Rubinson]] 22:51, 7 February 2013 (EST)''':Gabe, have you ever had any luck using the j5 program?
'''[[User:Max E. Rubinson|Max E. Rubinson]] 22:51, 7 February 2013 (EST)''':Gabe, have you ever had any luck using the j5 program?
 +
*'''[[User:Gabriel Wu|Gabriel Wu]] 01:02, 9 February 2013 (EST)''' J5 works reasonably well. It does what it advertises. It has a little overhead because you have to prepare about 9 files (some are optional) in order for it to run properly, but if you run a lot of Gibson reactions, it's worth the investment in time.
 +
*'''--[[User:Alvaro E. Rodriguez M.|Alvaro E. Rodriguez M.]] 00:43, 8 February 2013 (EST)''':I'm not sure, but thought that although not here, we should cover RNA synthesis somewhat.
*'''--[[User:Alvaro E. Rodriguez M.|Alvaro E. Rodriguez M.]] 00:43, 8 February 2013 (EST)''':I'm not sure, but thought that although not here, we should cover RNA synthesis somewhat.
 +
**'''[[User:Gabriel Wu|Gabriel Wu]] 01:05, 9 February 2013 (EST)''': RNA applications are pretty different from the ones discussed here. Also, many people who work with RNA will synthesize the RNA from DNA templates.  I'm not familiar with what the aptamer labs do, but that is another topic altogether.
 +
 +
 +
'''[[User:Kevin Baldridge|Kevin Baldridge]] 15:22, 11 February 2013 (EST)''':Maybe I missed something in today's talk about yeast recombineering, but how does one get the oligos into the yeast for recombination? On the part where he was talking about assembling 1kb pieces within yeast. I have never worked with yeast, are they able to take up linear DNA by itself?
 +
*'''[[User:Evan J WeaverIEvan Weaver]] 17:52, 14 February 2013 (CST)''':  I thought he said that he digested the cell wall (if it has one) with some enzyme (I don't remember what exactly).  I'd imagine that he would use something like heat shocking or electroporating the the PM, but I don't remember.
 +
== Assembling nonstandard bases ==
== Assembling nonstandard bases ==
*'''[[User:Catherine I. Mortensen|Catherine I. Mortensen]] 22:07, 6 February 2013 (EST)''': I noticed you mentioned that nontraditional bases could be assembled... I'm taking genetics now so I may learn about this soon but could you give an example when a nontraditional base would be useful? I assume a nontraditional base refers to another purine or pyrimidine?
*'''[[User:Catherine I. Mortensen|Catherine I. Mortensen]] 22:07, 6 February 2013 (EST)''': I noticed you mentioned that nontraditional bases could be assembled... I'm taking genetics now so I may learn about this soon but could you give an example when a nontraditional base would be useful? I assume a nontraditional base refers to another purine or pyrimidine?
**'''[[User:Yunle Huang|Yunle Huang]] 10:02, 7 February 2013 (EST)''': One example I found was 2-Aminopurine. 2-Aminopurine is a fluorescent nucleic acid analogues can be used in nucleic acid research. Since it pairs with both thymine and cytosine, it can also be used for mutagenesis. http://www.pnas.org/content/83/15/5434
**'''[[User:Yunle Huang|Yunle Huang]] 10:02, 7 February 2013 (EST)''': One example I found was 2-Aminopurine. 2-Aminopurine is a fluorescent nucleic acid analogues can be used in nucleic acid research. Since it pairs with both thymine and cytosine, it can also be used for mutagenesis. http://www.pnas.org/content/83/15/5434
**'''[[User:Benjamin Gilman|Benjamin Gilman]] 16:02, 7 February 2013 (EST)''': Unnatural bases are often used in synthetic DNA oligos to broaden the range of binding interactions or chemistry available (like what [http://www.somalogic.com Somalogic] has done with DNA aptamers).  Are there any applications where genes with synthetic, unnatural bases were used ''in vivo''?
**'''[[User:Benjamin Gilman|Benjamin Gilman]] 16:02, 7 February 2013 (EST)''': Unnatural bases are often used in synthetic DNA oligos to broaden the range of binding interactions or chemistry available (like what [http://www.somalogic.com Somalogic] has done with DNA aptamers).  Are there any applications where genes with synthetic, unnatural bases were used ''in vivo''?
 +
***'''[[User:Gabriel Wu|Gabriel Wu]] 01:10, 11 February 2013 (EST)''': I don't know of any "in vivo" applications for unnatural bases.
***'''[[User:Aurko Dasgupta|Aurko Dasgupta]] 20:34, 7 February 2013 (EST)''':So I'm guessing they handle stuff like promoter/transcription factor binding affinity? Unless you use some kind of unnatural tRNA as well, I doubt you'd be able get that stuff translated.
***'''[[User:Aurko Dasgupta|Aurko Dasgupta]] 20:34, 7 February 2013 (EST)''':So I'm guessing they handle stuff like promoter/transcription factor binding affinity? Unless you use some kind of unnatural tRNA as well, I doubt you'd be able get that stuff translated.
-
 
+
****'''[[User:Gabriel Wu|Gabriel Wu]] 00:27, 11 February 2013 (EST)''': In the article, the mention of non-traditional bases is simply an extension of the chemistry of DNA synthesis. Nucleotides all share a common deoxyribose phosphate backbone. The base that defines the nucleotide is not involved in the extension of the DNA molecule; therefore, any base can be incorporated into a growing chain (so long as it maintains the deoxyribose backbone).
 +
****'''[[User:Gabriel Wu|Gabriel Wu]] 00:27, 11 February 2013 (EST)''': However, I think it's interesting to note that researchers have studied how to synthesize XNA, or "xeno-nucleic acid," in order to understand the fundamental dynamics of the basic chemical nature of life and its ability to store information. They've actually made XNA polymerases that replicate XNA templates. While they haven't shown transcription of XNA into some RNA-equivalent molecule, the ability to have an XNA polymerase suggests that it is possible to create such a protein. [http://www.ncbi.nlm.nih.gov/pubmed/22517858]
 +
****'''[[User:Gabriel Wu|Gabriel Wu]] 01:09, 11 February 2013 (EST)''': Finally, there has been a  significant amount of work on unnatural amino acid incorporation. In this case, they modify a tRNA to incorporate an amino acid that is not its natural partner. This unnatural amino acid is then incorporated into the growing peptide chain. [http://www.ncbi.nlm.nih.gov/pubmed/2649980] While this modification is done to existing tRNAs that recognize natural codons (typically a stop codon), it is possible to imagine a scenario where a tRNA could be modified to recognize a transcribed XNA codon and used to incorporate an unnatural amino acid from XNA.
== iGEM connection ==
== iGEM connection ==
-
*'''[[User:Jeffrey E. Barrick|Jeffrey E. Barrick]] 00:35, 7 February 2013 (EST)''':Someone mentioned that CPEC was started as an iGEM project. Can you like to the relevant team website as part of the topic so that we can take a look at it?
+
*'''[[User:Jeffrey E. Barrick|Jeffrey E. Barrick]] 00:35, 7 February 2013 (EST)''':Someone mentioned that CPEC was started as an iGEM project. Can you link to the relevant team website as part of the topic so that we can take a look at it?
**'''[[User:Max E. Rubinson|Max E. Rubinson]] 22:43, 7 February 2013 (EST)''':I don't know if CPEC was started as an iGEM project, but [http://2009.igem.org/Team:Duke this group] at Duke (where the method was developed) used CPEC to assemble a metabolic pathway for the production of plastic polymers in ''E. coli''.
**'''[[User:Max E. Rubinson|Max E. Rubinson]] 22:43, 7 February 2013 (EST)''':I don't know if CPEC was started as an iGEM project, but [http://2009.igem.org/Team:Duke this group] at Duke (where the method was developed) used CPEC to assemble a metabolic pathway for the production of plastic polymers in ''E. coli''.
-
*'''[[User:Thomas Wall|Thomas Wall]] 20:33, 7 February 2013 (EST)''': http://2009.igem.org/Team:Duke
+
**'''[[User:Thomas Wall|Thomas Wall]] 20:33, 7 February 2013 (EST)''': http://2009.igem.org/Team:Duke
 +
**'''[[User:Gabriel Wu|Gabriel Wu]] 01:17, 11 February 2013 (EST)''':The paper on CPEC was published in July 2009. The only authors on the paper are the graduate student and PI involved in the iGEM team. [http://www.ncbi.nlm.nih.gov/pubmed/19649325] Considering the typical iGEM timeline and the publication date, as well as the lack of authorship for any of the undergraduate students, it's unclear if CPEC "started" as an iGEM project, but the students may have played some role in preparing it for publication.

Current revision

Gabriel Wu 23:39, 27 January 2013 (EST): Minor point: Companies like biomatik are not included on the genspace list. Is it necessary to make a wiki page with a "more" comprehensive list?

Gabriel Wu 00:49, 28 January 2013 (EST): This is a nice anecdotal piece of information that shows some concrete numbers for what DNA synthesis might have cost in June 2011. Unfortunately, it's not really appropriate for the main wiki page, but I post it here to live for eternity! Also, if anyone finds a more legitimate way of presenting this kind of data on the main page, then this comment might actually be useful. [1]

  • Aurko Dasgupta 20:51, 7 February 2013 (EST):I don't have any clear idea of the kind of budgets one should expect for making a 3000bp plasmid in lab, but getting 1 millimole of plasmid for ~$1200 actually sounds like a decent deal. Would it be reasonable to say that the labor and materials to make such a plasmid would amount to more than $1200, assuming a few mistakes along the way?
    • Max E. Rubinson 22:47, 7 February 2013 (EST):That's probably an issue between you and your boss.
    • Gabriel Wu 00:58, 9 February 2013 (EST): From people who do this kind of work, they tell me that assembling a 3 Kb plasmid takes about 3 full days of lab work. The cost of Gibson master mix from NEB is $154.00 for 10 reactions ($15 for one reaction). The cost of Phusion master mix is $170 for 100 reactions ($8 for 4 PCR reactions). Plates, media, and competent cells add a marginal cost (I would estimate generously no more than $20). Finally, oligoucleotides are about $15 a piece. This assembly would probably take about 8 long oligonucleotides ($160). Then finally, think about how much you make and add your 3 day cost. I estimate generously at about $300 for three days work. Add it all up, you get about $503. A two-fold difference at I think reasonably generous estimates. It becomes a question if whether your time is beter spent in other places and how quickly do you need the part.

Gabriel Wu 13:10, 4 February 2013 (EST): These are notes to myself. They help illustrate techniques in cloning or trends in DNA synthesis

Gabriel Wu 18:23, 4 February 2013 (EST): Added an introduction motivating the page. Explaining the need for "putting together" DNA.

  • Neil R Gottel 13:11, 7 February 2013 (EST):I was looking into Ginkgo Bioworks because this article at sciencenews mentioned they rely heavily on their own special CAD tools when designing their organisms, and found their blog. It hasn't been updated in a year, but their last post was talking about how slow and variable the turn-around times are for gene synthesis. They've got a chart showing the length of each order, and the time it took between when the order was placed, and when it was received. Some of their genes (or "synthons" as they call it, since some sequences are not whole genes) took more than two months to synthesize!
  • Gabriel Wu 00:44, 9 February 2013 (EST): So, this is where DNA assembly can fail. Depending on the number and stability of hairpins, the GC content, and whether or not there are inverted repeats (which leads to homologous recombination) in your sequence, synthesis companies can have a hard time making your DNA order. The advantage to using these services is that you don't have to deal with these things in the lab to construct them. However, synthesis companies have improved their ability to pre-screen these type of difficult sequences and often times will not accept the order at all. Gingko has actually moved away from ordering full assemblies and instead order oligonucleotides and then assemble the DNA in-house.

Thomas Wall 20:23, 7 February 2013 (EST): I had a formatting issue Gabe, You put CPEC and splice under gibson cloning, making it look like it is a subset of that when I think you mean for it to be a subset of overlap cloning.

Thomas Wall 20:31, 7 February 2013 (EST): This is a cool extension of CPEC I want to potentially start using in my lab (http://www.ncbi.nlm.nih.gov/pubmed/21293463)

Max E. Rubinson 22:51, 7 February 2013 (EST):Gabe, have you ever had any luck using the j5 program?

  • Gabriel Wu 01:02, 9 February 2013 (EST) J5 works reasonably well. It does what it advertises. It has a little overhead because you have to prepare about 9 files (some are optional) in order for it to run properly, but if you run a lot of Gibson reactions, it's worth the investment in time.
  • --Alvaro E. Rodriguez M. 00:43, 8 February 2013 (EST):I'm not sure, but thought that although not here, we should cover RNA synthesis somewhat.
    • Gabriel Wu 01:05, 9 February 2013 (EST): RNA applications are pretty different from the ones discussed here. Also, many people who work with RNA will synthesize the RNA from DNA templates. I'm not familiar with what the aptamer labs do, but that is another topic altogether.


Kevin Baldridge 15:22, 11 February 2013 (EST):Maybe I missed something in today's talk about yeast recombineering, but how does one get the oligos into the yeast for recombination? On the part where he was talking about assembling 1kb pieces within yeast. I have never worked with yeast, are they able to take up linear DNA by itself?

  • User:Evan J WeaverIEvan Weaver 17:52, 14 February 2013 (CST): I thought he said that he digested the cell wall (if it has one) with some enzyme (I don't remember what exactly). I'd imagine that he would use something like heat shocking or electroporating the the PM, but I don't remember.


Assembling nonstandard bases

  • Catherine I. Mortensen 22:07, 6 February 2013 (EST): I noticed you mentioned that nontraditional bases could be assembled... I'm taking genetics now so I may learn about this soon but could you give an example when a nontraditional base would be useful? I assume a nontraditional base refers to another purine or pyrimidine?
    • Yunle Huang 10:02, 7 February 2013 (EST): One example I found was 2-Aminopurine. 2-Aminopurine is a fluorescent nucleic acid analogues can be used in nucleic acid research. Since it pairs with both thymine and cytosine, it can also be used for mutagenesis. http://www.pnas.org/content/83/15/5434
    • Benjamin Gilman 16:02, 7 February 2013 (EST): Unnatural bases are often used in synthetic DNA oligos to broaden the range of binding interactions or chemistry available (like what Somalogic has done with DNA aptamers). Are there any applications where genes with synthetic, unnatural bases were used in vivo?
      • Gabriel Wu 01:10, 11 February 2013 (EST): I don't know of any "in vivo" applications for unnatural bases.
      • Aurko Dasgupta 20:34, 7 February 2013 (EST):So I'm guessing they handle stuff like promoter/transcription factor binding affinity? Unless you use some kind of unnatural tRNA as well, I doubt you'd be able get that stuff translated.
        • Gabriel Wu 00:27, 11 February 2013 (EST): In the article, the mention of non-traditional bases is simply an extension of the chemistry of DNA synthesis. Nucleotides all share a common deoxyribose phosphate backbone. The base that defines the nucleotide is not involved in the extension of the DNA molecule; therefore, any base can be incorporated into a growing chain (so long as it maintains the deoxyribose backbone).
        • Gabriel Wu 00:27, 11 February 2013 (EST): However, I think it's interesting to note that researchers have studied how to synthesize XNA, or "xeno-nucleic acid," in order to understand the fundamental dynamics of the basic chemical nature of life and its ability to store information. They've actually made XNA polymerases that replicate XNA templates. While they haven't shown transcription of XNA into some RNA-equivalent molecule, the ability to have an XNA polymerase suggests that it is possible to create such a protein. [2]
        • Gabriel Wu 01:09, 11 February 2013 (EST): Finally, there has been a significant amount of work on unnatural amino acid incorporation. In this case, they modify a tRNA to incorporate an amino acid that is not its natural partner. This unnatural amino acid is then incorporated into the growing peptide chain. [3] While this modification is done to existing tRNAs that recognize natural codons (typically a stop codon), it is possible to imagine a scenario where a tRNA could be modified to recognize a transcribed XNA codon and used to incorporate an unnatural amino acid from XNA.

iGEM connection

  • Jeffrey E. Barrick 00:35, 7 February 2013 (EST):Someone mentioned that CPEC was started as an iGEM project. Can you link to the relevant team website as part of the topic so that we can take a look at it?
    • Max E. Rubinson 22:43, 7 February 2013 (EST):I don't know if CPEC was started as an iGEM project, but this group at Duke (where the method was developed) used CPEC to assemble a metabolic pathway for the production of plastic polymers in E. coli.
    • Thomas Wall 20:33, 7 February 2013 (EST): http://2009.igem.org/Team:Duke
    • Gabriel Wu 01:17, 11 February 2013 (EST):The paper on CPEC was published in July 2009. The only authors on the paper are the graduate student and PI involved in the iGEM team. [4] Considering the typical iGEM timeline and the publication date, as well as the lack of authorship for any of the undergraduate students, it's unclear if CPEC "started" as an iGEM project, but the students may have played some role in preparing it for publication.
Personal tools