Matt Gethers: Difference between revisions

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I'm currently a sophomore in course 20. When I'm behaving myself, I'm allowed to work in the [[Endy Lab]] at [http://mit.edu MIT]. I will be documenting my UROP projects [[Endy:Translation demand | here]]. I will put my 20.109 work directly below.
I'm currently a sophomore in course 20. When I'm behaving myself, I'm allowed to work in the [[Endy Lab]] at [http://mit.edu MIT]. I have links to my UROP projects and 20.109 work directly below.


==20.109 Work==


===M13 Engineering Ideas===
==UROP Projects==
 
[[/20.380 HIV Project|20.380 HIV Project]]
 
[[/Articles of General Interest| Articles of General Interest]]


{| {{table}}
[[/Articles to Read|Articles to Read]]
| align="center" style="background:#f0f0f0;"|'''Gene'''
| align="center" style="background:#f0f0f0;"|'''Modification'''
|-
| X||Extract from gene II.
|-
| II||Extract gene X.
|-
| V|| Add some base pairs between V and VII to allow for a restriction site.
|-
| VII||Separate from gene IX.
|-
| III||Change the GTG to ATG Start?
|-
| VI|| Add some base pairs between III and VI and VI anda I to allow for restriction sites.
|-
| I||Separate from genes IV and XI.
|-
| XI||Separate from genes I and IV.
|-
| IV||Separate from genes I and XI.
|-
| M13 ORI 1 & 2|| Are two ORIs necessary? If so, can they be consolidated?
|-
| KanR|| Possible to remove bps 6600 - 7100 upstream of KanR? Does this DNA have functional significance?
|-}


[[Endy:Translation demand | Translation Demand]]


Extractions, separations, etc. include ensuring that each gene has its own promoter, RBS, terminator, and any other pertinent regulatory sequences.
[http://bcanton.private.openwetware.org/wiki/Lab_Notebook/Matt/Mitochondria_Project Mitochondrial Engineering]


In addition to separating the genes, it would also be wise to develop or utilize/modify an existing system like [http://parts.mit.edu/registry/index.php/Main_Page Biobricks] to build the genome so that convenient restriction sites exist between the genes so they can be easily removed or replaced.
[[/CRI, Thailand| CRI Thailand]]


I used [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/sequences/m13ko7.txt the following annotation] to locate and design changes to the genome.
[[/Matt's Stanford Freezer Stocks|Matt's Stanford Freezer Stocks]]


[[/OWW Syntax| OWW Syntax]]


[[/Eloranta Ideas|Eloranta Ideas]]


{| {{table}}
==20.109 Work==
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''2.27.07 Ligation and Transformation Data'''
| align="center" style="background:#f0f0f0;"|''''''
|-
| align="center" style="background:#f0f0f0;"|'''DNA Ligation Sample'''
| align="center" style="background:#f0f0f0;"|'''Expected Number of Transformants'''
| align="center" style="background:#f0f0f0;"|'''Observed Number of Transformants'''
|-
| Control Plasmid||Many||1184
|-
| BKB with cocktail but no ligase||0||0
|-
| BKB with ligase and cocktail||0||6
|-
| BKB with insert and ligase and cocktail #1||A few||0-1
|-
| BKB with insert and ligase and cocktail #2||A few||5
|-}


===2.27.06 Ligation and Transformation Data===
*[[User:Mgethers/M13 Engineering Ideas|M13 Engineering Ideas]]


We didn't determine the concentration of the prepped DNA from the ligation reactions, so we cannot calculate the transformation efficiency of these transformations. We do know, however, that 5 ng of vector was used to transform the control plasmid, so efficiency = (1184 colonies)/(5*10^-3 micrograms of DNA)= 5.92*10^6 colonies/microgram of DNA.
*[[User:Mgethers/2.27.06 Ligation and Transformation Data|2.27.06 Ligation and Transformation Data]]


The control plasmid transformation serves as a positive control. It should work. If this transformation didn't work (along with some or all of the others), we might suspect an issue with the competency of the cells or with the transformation process. The appearance of colonies on this plate, however, demonstrates that this is not the case. The cells are indeed competent and our protocol worked.  
*[[User:Mgethers/2.27.09 Refactoring Work|2.27.09 Refactoring Work]]


The backbone with and without ligase mixed with the killcut cocktail serve as negative controls. They should not work. If a significant number of colonies were to show up on these plates, this would first mean that the restriction endonucleases weren't cutting. This could be because they had expired or because the sites no longer existed in the DNA. Assuming the latter for the ligated vector, this would mean that the vector sequence was somehow modified during the initial digest. For the linearized vector, the appearance of colonies would suggest that the DNA somehow became ligated and lost restriction sites, or the cell accepted cut vector.  
*[[:Image:4.3.07_Primer_Design_Team_Purple.doc|4.3.07_Primer_Design_Team_Purple.doc]]


The controls do not provide a definitive diagnosis of the problem should the experimental ligations fail, but they provide starting points for the inquiry into the failure.
*[[User:Mgethers/Research Proposal|Research Proposal]]


===2.27.09 Refactoring Work===
==Class projects==
My refactoring work generally followed the guidelines set out by Chan et al's "Refactoring T7". I started by designing [[http://parts.mit.edu/registry/index.php/Part:BBa_M31530| BBa_M31530]] directly from the germane promoters, RBSs, and ORFs. As an exercise in abstraction, I then started defining more complex functional units. First I defined a gene with its transcriptional and translational regulatory regions as a part. In this way, I don't have to think about how the gene is expressed so much as the end result. An example is [[http://parts.mit.edu/r/parts/partsdb/part_info.cgi?part_name=BBa_M31532| Gene X (BBa_M31532)]]. By the same token, I was able to create functional units of several genes related by their functions. For example, I created [[http://parts.mit.edu/r/parts/partsdb/part_info.cgi?part_name=BBa_M31538| BBa_M31538]] which is made of all the genes responsible for gene amplification of M13K07 while in a host and I made [[http://parts.mit.edu/r/parts/partsdb/part_info.cgi?part_name=BBa_M31540| BBa_M31540]] for phage coat proteins. Within each of these, I attempted to address the possibility of intergenetic cross-talk by introducing transcriptional terminators between the genes [[http://parts.mit.edu/registry/index.php/Part:BBa_B0015| BBa_0015]]. From my work with Heather Keller, I also know that hair pins (5' and 3' UTRs) can play a role in stabilization of DNA and posit that they may be helpful in preventing recombination events. I was unable to locate hairpins as parts in the registry, but I would like to add them and make us of them in my refactored M13K07.
[[/20.310 Term Paper| 20.310 Term Paper]]


Here are a few tables of my work.  
==20.310 Project==


{| {{table}}
<biblio>
| align="center" style="background:#f0f0f0;"|'''Gene'''
#Galkin pmid=17449671
| align="center" style="background:#f0f0f0;"|'''Modification'''
#Carragher pmid=3351926
|-
#Carragher2 pmid=3351927
| X||Extract from gene II.
#Carragher3 pmid=3351930
|-
#Turner pmid=12638863
| VII||Separate from gene IX.
#Wang pmid=11812133
|-
</biblio>
| VIII||Separate from gene IX.
|-}


{| {{table}}
Treatments exist for preventive and palliative treatment of sickle cell episodes, but no treatment has yet been developed to mitigate the effects of an attack once it has started. We need a way of dealing with Hbs fibers once they have polymerized. We feel we can develop a treatment for an ongoing attack by addressing the biomechanical basis of the pathology.
| align="center" style="background:#f0f0f0;"|'''Abstraction (Functional Unit)'''
| align="center" style="background:#f0f0f0;"|'''Components of Functional Unit'''
|-
| The Regulatory/Coding Region||Strings of DNA
|-
| The Gene||The Promoter, RBS, and ORF
|-
| A Portion of the Life Cycle||The genes responsible for a certain part of the life cycle
|-}

Latest revision as of 05:22, 18 February 2009

I'm currently a sophomore in course 20. When I'm behaving myself, I'm allowed to work in the Endy Lab at MIT. I have links to my UROP projects and 20.109 work directly below.


UROP Projects

20.380 HIV Project

Articles of General Interest

Articles to Read

Translation Demand

Mitochondrial Engineering

CRI Thailand

Matt's Stanford Freezer Stocks

OWW Syntax

Eloranta Ideas

20.109 Work

Class projects

20.310 Term Paper

20.310 Project

  1. Galkin O, Pan W, Filobelo L, Hirsch RE, Nagel RL, and Vekilov PG. Two-step mechanism of homogeneous nucleation of sickle cell hemoglobin polymers. Biophys J. 2007 Aug 1;93(3):902-13. DOI:10.1529/biophysj.106.103705 | PubMed ID:17449671 | HubMed [Galkin]
  2. Carragher B, Bluemke DA, Gabriel B, Potel MJ, and Josephs R. Structural analysis of polymers of sickle cell hemoglobin. I. Sickle hemoglobin fibers. J Mol Biol. 1988 Jan 20;199(2):315-31. DOI:10.1016/0022-2836(88)90316-6 | PubMed ID:3351926 | HubMed [Carragher]
  3. Bluemke DA, Carragher B, Potel MJ, and Josephs R. Structural analysis of polymers of sickle cell hemoglobin. II. Sickle hemoglobin macrofibers. J Mol Biol. 1988 Jan 20;199(2):333-48. DOI:10.1016/0022-2836(88)90317-8 | PubMed ID:3351927 | HubMed [Carragher2]
  4. Carragher B, Bluemke DA, Becker M, McDade WA, Potel MJ, and Josephs R. Structural analysis of polymers of sickle cell hemoglobin. III. Fibers within fascicles. J Mol Biol. 1988 Jan 20;199(2):383-8. DOI:10.1016/0022-2836(88)90322-1 | PubMed ID:3351930 | HubMed [Carragher3]
  5. Jones CW, Wang JC, Ferrone FA, Briehl RW, and Turner MS. Interactions between sickle hemoglobin fibers. Faraday Discuss. 2003;123:221-36; discussion 303-22, 419-21. DOI:10.1039/b207388a | PubMed ID:12638863 | HubMed [Turner]
  6. Wang JC, Turner MS, Agarwal G, Kwong S, Josephs R, Ferrone FA, and Briehl RW. Micromechanics of isolated sickle cell hemoglobin fibers: bending moduli and persistence lengths. J Mol Biol. 2002 Jan 25;315(4):601-12. DOI:10.1006/jmbi.2001.5130 | PubMed ID:11812133 | HubMed [Wang]

All Medline abstracts: PubMed | HubMed

Treatments exist for preventive and palliative treatment of sickle cell episodes, but no treatment has yet been developed to mitigate the effects of an attack once it has started. We need a way of dealing with Hbs fibers once they have polymerized. We feel we can develop a treatment for an ongoing attack by addressing the biomechanical basis of the pathology.

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