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author | Bryan Bishop <kanzure@gmail.com> | 2018-03-14 08:38:50 -0500 |
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committer | Bryan Bishop <kanzure@gmail.com> | 2018-03-14 08:38:50 -0500 |
commit | ccb4c71919ac7a0121bf0c558fef6b1f4d3bb9a1 (patch) | |
tree | 726c5325ac8d0a7e75724f73561093ce68acae1a | |
parent | 1961d5e43e1492511c6c924db923f83aead49699 (diff) | |
download | diyhpluswiki-ccb4c71919ac7a0121bf0c558fef6b1f4d3bb9a1.tar.gz diyhpluswiki-ccb4c71919ac7a0121bf0c558fef6b1f4d3bb9a1.zip |
more hgp-write 2017 transcripts
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diff --git a/transcripts/hgp-write/2017-05-09/biologically-inspired-engineering-of-microorganisms.mdwn b/transcripts/hgp-write/2017-05-09/biologically-inspired-engineering-of-microorganisms.mdwn new file mode 100644 index 0000000..186cc1e --- /dev/null +++ b/transcripts/hgp-write/2017-05-09/biologically-inspired-engineering-of-microorganisms.mdwn @@ -0,0 +1,25 @@ +Biologically inspired engineering of microorganisms + +Jay Konieczka, enEvolv + +<https://www.youtube.com/watch?v=VlWj9gfpS5Q&t=0s&list=PLHpV_30XFQ8RN0v_PIiPKnf8c_QHVztFM&index=55> + +We engineer microbes to produce products both together with partners and our own proprietary targets. We have technology inside the cell that allows us to build and screen billions of genomic designs in a matter of a month. This is without heavy investment in automation. + +The challenge in engineering microbes is the complexity of biology. We're all familiar with the design-build-test-analyze loop. We all know this. As you iterate through this process, you have to be very aware now of-- we're beyond the scope of optimizing a single pathway. We're looking at a whole organism. It requires a tremendous amount of throughput. Our platform does exactly that. + +We have variation technology which delivers mutations in a massively parallel fashion. Then we screen the cells on a high-throughput selection mechanism. And then we analyze, characterize and learn from them, and then we influence the net xesign phase. + +The variation technology is actually MAGE. It allows us to deliver massively parallel mutations single point mutations and arrays of point mutations, insertions, deletions, at multiple loci in the genome simultaneously. Our cultures have every strain in the culture that are unique from each other. So it's combinatorial and you have to screen through the haystack. + +We have allosteric transcription factors that we engineered to recognize specific and desired target molecules. That's how we do sscreening. In some cases, within weeks, we can turn around a sensor or a panel of sensors that respond in different fashions to the production of our target molecules. These drive gene expression. We use reporters or selection markers, like GFP, and those strains that produce more, we can immediately see that in the population and select for that. + +The sweet spot is right there at the build and test cycle. If you were to do this with robotics, then you would need like $10M in equipment just to get to 10,000 designs per month. But for us, we can do 1 billion strains per month. This scales linearly. We could do this with traditional automation but it's not necessary. + +Our tech is portable. We are in multiple species. Raising series B round. Because of high probability of success, we're taking on projects that we wouldn't do with traditional methods. As a result we're growing and raising our series B round of funding. + +# Q&A + +Q: .. doing hte permutations all together in one tube. When you're trying to optimize production of something that can float out of the cell and into other cells, can you identify winners? + +A: There's a couple of ways we approach that. At the end of the day, when you want to look at an end product that is diffusable, you have to separate the cells, like microencapsulation. There's other tactics sometimes like engineering a sensor for an upstream metabolite that we make sure doesn't leak at least not quickly, and then optimize for that. diff --git a/transcripts/hgp-write/2017-05-09/musebio.mdwn b/transcripts/hgp-write/2017-05-09/musebio.mdwn new file mode 100644 index 0000000..9e8f6d9 --- /dev/null +++ b/transcripts/hgp-write/2017-05-09/musebio.mdwn @@ -0,0 +1,31 @@ +Genome scale engineering + +Kevin Ness, CEO, Muse Bio + +bd@musebio.com + +<https://twitter.com/kevin_dean_ness> + +<https://www.youtube.com/watch?v=qeb0KC9bs98&t=0s&list=PLHpV_30XFQ8RN0v_PIiPKnf8c_QHVztFM&index=56> + +Single cell writing + +Sold a digital PCR company to Agilent(?). Started 10x genomics. Those were focused on better tools to read the genome. Now the focus is better tools to write the genome. I can't do this alone, I am a tool builder not a tool user. We are inserting barcodes so that you can track each cell. We started in 2015 from U of Colorado. 6M series A. 23M series B from lead investors in intel and illumina. We just announced founder/chairman of illumina joining our board. We want to write the genome. Expected commercial launch end of 2018. + +Single cell writing with trackable multiplex editing + +Some of this was published in Nature Biotech brand name will be ForgeCraft biochemistry. "Create" (?). + +96 well plate is limited. You are limited to the number of wells. If you want to do an edit with a guide RNA and so on, and you need more wells. Whether you need a library of guide RNA, or a library of homology arms, to try to increase the number of edits per experiment, you are limited by the plate. Don't think of using the well as the partition. Let's use the cell as the partition. Using the cell as the partition, you can put 100k well plate in a normal 96 well plate. How do you get the reagents colocalized always going into the right cell? + +So they are covalently attached-- gRNA and homology arm are combined. Also, you can trakc it, so there's a barcode attached using biochemistry. + +We rationally designed every variant for the FolA enzyme. We screened activity to find viable variants. 100x reduction in cost, 200x increase in activity, >3140 mutants, novel mutations identified. This is a well studied protein but we found novel variants because we're doing full saturation of the enzyme, literally every combination of the amino acid sequence. So let's jump from one gene and go to 19 genes. + +Here you're now studying a pathway. We wanted to increase the lysine production. We got 100x increase in variation space with 10x reduction in time. There was a 1000x increase in growth. 16300 variants, 19 genes. We are doing mass spec verification. + +We also did a network with 177 genes. We made 75,093 variants. 64x increase in antibiotic resistance was discovered. + +ForgeCrafter (software), Forge (instrument), and Forge kits (reagents). Fully automated plasmid and single cell gene writing with barcodes. + + diff --git a/transcripts/hgp-write/2017-05-09/synthetic-regulatory-genomics.mdwn b/transcripts/hgp-write/2017-05-09/synthetic-regulatory-genomics.mdwn new file mode 100644 index 0000000..9f05cef --- /dev/null +++ b/transcripts/hgp-write/2017-05-09/synthetic-regulatory-genomics.mdwn @@ -0,0 +1,52 @@ +Dark matter of the human genome: Synthetic regulatory genomics + +Matt Maurano, NYU Langone Medical Center + +<https://www.youtube.com/watch?v=xvlkdTgqo3A&t=0s&list=PLHpV_30XFQ8RN0v_PIiPKnf8c_QHVztFM&index=13> + +This is the slide for mammalian gene regulation. This alludes to the complexity of the regulatory genes, and gives us all the interesting biology and medicine that you will hear about. What's going on in this slide is that there are chromatid fibers, wrapping around some DNA. There are transcription factors binding over here. The binding of these TFs is specified by sequence, but this sequence plays out in an epigenetic context and has many differences in effects from one cell type to another. The point of having all this up here is to give you a sense of genomic content. If you move these regulatory elements around, and people have done this in a small scale in the past, so this provides complication for study gene regulation but also an opportunity for this group here. + +Originally mapping regulatory DNA was based on accessibility. These days we can do this on a genome-wide scale. You see tracks like this, where each peak reppresents a particular regulatory element. These are transcription factors- if you go look in different cell types, there's a great variety of regulatory landscape for cell types. These are things like promoters, enhancers and other regulatory elements. They have been mapped in great depth over the last 5 years by a large national consortium. You can see here a list of a huge variety of cell and tissue ypes for which data is available publicly on the internet identifying regulatory elements. + +An individual cell type might be 1% of the genome, represented by regulatory elements. Because they are specific, there might be up to 4 million regulation elements in the genome. There's a lot of material out there. + +What do these elements do? + +We use terms like promoters and ehancers. But it doesn't really scale to what we see today in terms of the genomics movement. You can look at the binding of multiple different TFs, repressors, etc. There's a lot of stuff going on at each loci. Why hasn't this been answered yet? + +Maurano et al Science 2012 Mapping human disease and trait-associated variation by genome-wide association studies (GWAS) + +For common diseases and traits that many of us are interested in, .. the majority of the hits lie in non-coding DNA. 5% of the hits are landing in protein-coding regions. The rest are landing in non-coding DNA and they are highly concentrated in these regulatory elements. So this presents a rather depressing picture at first glance for medicine. + +Why is this a hard problem to tackle? + +There are two prospective ways-- one is to study regulatory variation in its endogenous context, in the first it's engineering variation, and this has taken many decades but it has been accelerated by using nucleases. This gives you a really high degree of control. You can extend this to many sites, with some technical complications but there are some limitations as to what you are able to do. You want to start doing multiple changes, which are getting harder. + +The other approach is to look at natural variation. You can do genome profiling to find gene expression traits. This gives you efficient yield. We've done some work using .. if you will.. we've been able to build local models, and study sequence variation effects. + +Endogenous approaches to study regulatory variation + +We don't have reporter assays able to study regulatory variations for all the uestions we might like to ask. We could classify these by reporter size, like plasmids that are smaller than 5 KB, or BACs which are 100-300 kb, or YACs which are 100-1000 kb. You could clasiffy by integration like in vitro, transient, stable (random), site-directed single-copy. And you can clasisfy by scale- traditional single reporter, or multiplexed. + +You start going back and looking at locus and loci at large scale. We're interested in pushing this forward, a group of us at NYU as well as other places. This is a quick overview orf orur strategy. There's a BAC vector in which we can integrate a large segment of DNA up to a few kilobases, containing a gene of interest, and at places of interest of hypersensitive sites, that lets us place modules, to focus our attention on elements that are important and begin to scale in-depth analysis. + +The problem is delivering these guys... so we do scarless single-copy, site-specific integration and use counter-selection. Ultimately the interesting part is going to be what are you going to do to do profiling, like RNA profiling via rt-PCR, chromatin accessibility via capture DNAse-seq, and chromating capture via something-C. + +This opens up a lot of problems to address: multi-edited synthetic haplotypes, cross species function, gene fusions, chromosomal rearrangements, position effect variations. + +You can make single substitutions, pairwise substitutions. We scan through single knockouts, we do this rapidly and base on the results we test double knockouts as well as other types of changes. + +We have been working on an alpha globin locus in collaboratorion with Hay lab, see Hay et al. Nat Genet. 2016. They did this in mice, it has been informative for locus control around these genes, and we can go well beyond previous studies by increasing the scale. + +Maurano labMegan Hogan, Jesper Maag, Nick Vulpescu, Maia Stoicvici, and collaborators like Boeke lab, LIMS/automation like Sergei German, Andrew Martin, Henri Berger, Vincent German, and Doug Higgs, Tim Niewold, Aravinda Chakravarti. + +# Q&A + +Q: When you make heterozygtes, do you have problems analyzing hypersensitive regions? + +A: We have done a lot of work looking at natural variation. The advantage of doing it this way is that you can do point variants as markers. Some of the first ways are going to be copying-- we can go back in and put in markers so we can distinguish them. + +Q: Perfect example is the advantage of synthetic over natural. + +A: That's true. + diff --git a/transcripts/hgp-write/2017-05-09/technology-for-the-construction-of-synthetic-bacteria.mdwn b/transcripts/hgp-write/2017-05-09/technology-for-the-construction-of-synthetic-bacteria.mdwn new file mode 100644 index 0000000..242982f --- /dev/null +++ b/transcripts/hgp-write/2017-05-09/technology-for-the-construction-of-synthetic-bacteria.mdwn @@ -0,0 +1,82 @@ +Technology for the construction of synthetic bacteria + +Kazuhito Tabata + +University of Tokyo + +<https://www.youtube.com/watch?v=yGMqyKiAKVg&t=0s&list=PLHpV_30XFQ8RN0v_PIiPKnf8c_QHVztFM&index=18> + +# Introduction + +I'd like to thank the organizers for this opportunity. In the past, I was involved in ImPACT, impulsing paradigm change through disruptive technologies program. The aim of our program -- bio-innovation through artificial cell reactor. Using a microreactor array, here. The project can be broken into 3 subprojects. We aim to enhance the ... of a single-molecule reaction. + +Create to synthesize the function of protein, and ... finally, we aim to proliferate the.. such as is a ... and through.. and inside of our reactor. Today my attention will be on the proliferate project. + +Masayuki Su'etsugu, Rikkyo University and myself. Genome replication and gene annealing by a cell-free in vitro cloning method. And my project focus was on Creation of artificial cells by fusing bacteria and microdevices-- technologies for genome exchange. + +# Cell-free cloning of large circular DNA + +Current popular techniues about 3 days. Furthermore, we developed a ... gene fragment assembly, we lead to, ... this approach reduces exposure. We also, because we demonstrate it with a general application, we prove that it can handle large DNA. In his slide, I am showing in vitro reconstitution of replication "cycle" of ecoli chromosome. Replication cycle reacion - RCR. Auonomous repetition of the oriC-replication cycle at 30 degrees celsius. + +# DNA amplificiation in RCR + +... + +# Amplification of several hundred kb DNA by RCR as covalently closed circles + +It's possible to do this up to 200 kb in size. We have achieved 350 kb. We are now working on megabase size DNA. + +# Gene assembly method + +Two-step reaction to produce circular DNA from multiple fragments, which are ligated via overlap ends, and then RCR. The assembly step is a 30 minute isothermal reaction. Specific amplification of circular assembly. Even if the yield of the ligation reaction is low, and cloning can be quite high + +# Cell-free in vitro cloning kit + +Scheduled for free distribution just ask: +kazuhito@nojilab.t.u-tokyo.ac.jp + +# Creation of artificial cells by fusing bacteria and microdevices + +Introductio nof synthetic mateiral into bacteria, fuse the device and bacteria to prepare an artificial cell. + +Membrane fusion technique. + +# Arrayed lipid bilayer chamber (ALBiC) + +Bilayer production efficiency is over 99%. Encapsulate dye in bilayer chamber. We reconsittute alpha-hemolsyin in bilayer chamber. Passive transport was observed. Next, we confirm that the bilayer membrane had .. function. We observe.. alpha-hemolysin in bilayer chamber. We can verify it by difufsion of fluorescent dye. It decreases over time. We confirmed that we did not impair it. + +# Fusion of EP and ALBiC (hybrid cell) + +I'd like to show you a vide oof the fusion. In some hybrid cells, the addition of ATP increased the synthesis of GFP. + +This graph shows the fluorescence intensity of the hybrid cells. The x-axis is a presents fluorecence from the beginning of the experiment, y-axis is 3 hour interval. + +# Beta-gal expression in DNA plasmid + +We confirmed beta-gal expression in plasmid. SPiDER. + +# Does regeneration occur in hybrid cells? + +You can see the fusion. And here you see the hybrid cell engage with the reactor like this. In this red circle, you can see the glow spike object like this, and here. Finally, in this movie, you can see the small part in the hybrid cell. This observation demonstrates regeneration in the microdevice. + +# Conclusion + +New system for replication and propagation of the ecoli genome. + +Cell-free cloning gene annealing in the absence of ecoli + +Successful integration of ecoli and microdevice + +Bacteria regeneration in hybrid cell (although only slightly) + +# Q&A + +Q: In vitro cloning kit, can it replace in vivo cloning? Have you shown you can go to a limiting dilution of single plasmids? With in vivo cloning you get one plasmid per cell. + +A: Contamination is a problem, yes. Difficult to remove the contamination. + +Q: ... what's the difference between volume in incoming ecoli cells, and the bioreactor cells? It's surprising you get that much expression, it's going to limit your ability to produce hybrid cells. + +A: The bioreactor is tiny. It's roughly the same volume. Ecoli size is about 3 or 4 micrometer diameter. My device ... just.. + +Thank you. |