Endy:BAC Subgroup: Difference between revisions

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*This week
*This week


==Week of 7/17/17==
==Week of 8/7/17==


'''Alec'''
'''Alec'''


Last week - Got a working prototype of the program working and chose a standard vector for protein purification (pET28+). Showed that the plasmid could be linearized with the primers that were ordered (they seemed iffy).
Last week - Tested the prototype of the program and generated multiple (10) primers for PURE parts. Tested the PCR once and all failed; will retry to see if failure is due to primer design or experimental setup. Also made a list of reactions that occur in PURE in preparation to build the PURE model.
 
This week - Re-PCR the 5 components that I have designed via the program and see if amplification occurs. Build the model of PURE based on the Katy Wei thesis to be presentable at the Build-A-Cell meetup in Pasadena.


This week - Scale up the program and make primers for all components that we have now once protein purification is shown to work with the vector we are using (waiting on Eric for results). Start cloning the constructs out of ''E. coli'' and put them in vectors for protein purification. Figure out how to handle modeling/find out what to present for the Caltech meeting. Though at the risk of sounding like someone who is all talk no action, I'd like to have a joint meeting with all interested in the modeling aspect of the project to figure out what is tractable for the Caltech meeting and REU as well as what would be most useful to researchers working with the PURE system currently.


'''Anton'''
'''Anton'''
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'''Cynthia'''
'''Cynthia'''
*''Reconstitution of E. coli RNA polymerase in PURE'' - PCR, Gibson, transformation, and miniprep of RNA polymerase subunit genes were completed. DNA has been sent to Elim Bio for sequencing.
*''Reconstitution of E. coli RNA polymerase in PURE'' - All templates for 5 subunits have been cloned into plasmid and linear templates, reconstituted E. coli RNAP with plasmid templates works with nanoluciferase assay
*''Characterization of E. coli RNA polymerase promoter library'' - E. coli holoenzyme and promoter DNA constructs have come in. First PURE experiment with J23119 and J23100 promoters and holoenzyme will be completed Tuesday
*''Characterization of E. coli RNA polymerase in PURE'' - Ran successful PURE reactions with nanoluciferase and GFP linear templates, but mRFP-Spinach reporter is still non-functional. Will debug PCR for mRFP-Spinach linear templates with 25-bp handles.
*''Liposome formation'' - First liposome experiment will be completed Tuesday or Wednesday
*''Liposome formation'' - Working on reconstitution of "outer solution"/solution A of PURE.
*''Protein purification'' - Will learn the procedure from Eric this week.




'''Eric'''
'''Eric'''
*''Make corrections to initiator tRNA structure and order ultramers'' - Reading old tRNA papers to figure out potential failure mode for nonfunctional initiator tRNA
* ''Purify IF1 and IF2'' - still cloning, but have ValS + others to test
*''Purification of fmt enzyme'' - Underwent purification, will run on gel to check if successful
* ''Clone 4 vectors for PURE proteins'' - Have 8 in progress
 
* Clone and purify out 6 more PURE proteins
* Read into potential maturation enzymes necessary for the maturation of the initiator tRNA


* Continue purification of PURE components
* Reconstitute Sol A and verify using commercial PURE kit


'''Keoni'''
'''Keoni'''
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'''Vicky'''
'''Vicky'''
*SUMMER PROJECT: rebuilding the 50S subunit of the ribosome. This includes synthesizing its different parts: r-proteins, coenzymes, 5S rRNA and 23S rRNA, the latest being the most challenging.
*SUMMER PROJECT: reconstructing the 50S subunit of the ribosome. This includes synthesizing its different parts, expressing them in PURE, and coupling it with native 30S to text for functionality.
*''Put together latest work on ribosome biogenesis'' - putting together the latest information available on ribosome biogenesis
*''Put together latest work on ribosome biogenesis'' - constantly looking for updates
*''Synthesizing primers for ribosomal genes'' - Alec is working to create a program that allows us to create primers quickly and effectively since we will be creating a large quantity of primers
*''Synthesizing primers for ribosomal genes'' - working with Alec to create a program that allows us to create primers specific to create our vector (would need it for over 110 genes)
*''Other'' - Learning wetlab skills: PCR, Primer design, Protein Purification, Gibson assembly, Bacterial transformation, Competent cell preparation
 
*"This week": continue to purify more ribosomal components into vector and get them to express in pure + look for other labs that se an automated method of cloning as opposed to monocloning

Latest revision as of 09:17, 7 August 2017

BAC Weekly Task List

List of planned work for the week for each individual on team

Suggested format:

  • Last week - Progress
  • This week

Week of 8/7/17

Alec

Last week - Tested the prototype of the program and generated multiple (10) primers for PURE parts. Tested the PCR once and all failed; will retry to see if failure is due to primer design or experimental setup. Also made a list of reactions that occur in PURE in preparation to build the PURE model.

This week - Re-PCR the 5 components that I have designed via the program and see if amplification occurs. Build the model of PURE based on the Katy Wei thesis to be presentable at the Build-A-Cell meetup in Pasadena.


Anton


Conary


Cynthia

  • Reconstitution of E. coli RNA polymerase in PURE - All templates for 5 subunits have been cloned into plasmid and linear templates, reconstituted E. coli RNAP with plasmid templates works with nanoluciferase assay
  • Characterization of E. coli RNA polymerase in PURE - Ran successful PURE reactions with nanoluciferase and GFP linear templates, but mRFP-Spinach reporter is still non-functional. Will debug PCR for mRFP-Spinach linear templates with 25-bp handles.
  • Liposome formation - Working on reconstitution of "outer solution"/solution A of PURE.
  • Protein purification - Will learn the procedure from Eric this week.


Eric

  • Purify IF1 and IF2 - still cloning, but have ValS + others to test
  • Clone 4 vectors for PURE proteins - Have 8 in progress
  • Continue purification of PURE components
  • Reconstitute Sol A and verify using commercial PURE kit

Keoni

  • Last week: Collection of materials for testing Bacillus subtilis minipreping and storage. Tested conjugation with Vibrio natriegens.
  • This week: Preparing DNA synthesis sequences.

Maria

Last week:

  • Successfully transformed the conjugative plasmid pRK24 into recipient strain MG1AC53.
  • Next step on this confirming the presence of pRK24 with gel.

This week:

  • Read literature on no-SCAR system with the purpose of replacing the oriT-kan casette with negative selection marker SacB on pRK24. Design appropriate sequences to carry out this experiment. This would be done to avoid back-conjugation of the genome from the recipient to the donor.

Vicky

  • SUMMER PROJECT: reconstructing the 50S subunit of the ribosome. This includes synthesizing its different parts, expressing them in PURE, and coupling it with native 30S to text for functionality.
  • Put together latest work on ribosome biogenesis - constantly looking for updates
  • Synthesizing primers for ribosomal genes - working with Alec to create a program that allows us to create primers specific to create our vector (would need it for over 110 genes)
  • "This week": continue to purify more ribosomal components into vector and get them to express in pure + look for other labs that se an automated method of cloning as opposed to monocloning
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