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Team:Johns Hopkins/Notebook
From 2008.igem.org
Contents |
Groups
iGEM Groups 1.0
iGEM Groups 2.01
iGEM Groups 2.02
Important reminders and notes
[Make general comments here, so they don't get lost in peoples e-mail boxes]
July 11: Primers for group 1 were delivered yesterday.
July 11: Lab meeting at 7:30PM in the lab to go over miniprep protocol.
July 15: Lab meeting at 6:30PM with Jessica. Have status reports ready.
Bring labtop if you can.
July 17: Restriction Digest/Sequencing Preparation (with James) 6:00PM.
July 21: 6:00PM or 6:30PM Lab meeting with Jessica. Have status reports ready.
July 29: 7:00PM Lab Meeting. Meet in Conference Room across from lab. Have Status Reports ready
Aug. 05: 7:00PM Lab Meeting. Meet in Conference Room across from lab. Have Status Reports ready.
Aug. 12: 7:00PM Lab Meeting. Have Status Reports Ready.
Journal Club Topic: Fluorescent Proteins; James and Ingrid.
Aug. 19: 7:00PM Lab Meeting. Have Status Reports Ready.
Journal Club Topic: Yeast Mating Pathway/MAP Kinase Pathway ; Jasper and Tejas
Aug. 26: 7:00PM Lab Meeting. Have Status Reports Ready!
Journal Club Topic: S. cerevisiae Promoters; Allison and Nate
EVERY TUESDAY 7 PM LAB MEETING! Mudd 120
September: Do well in classes. Do what you can for the team when you can.
October: Do well on midterms. Good Luck!!
Oct. 28, 2008: 7:00 PM Lab Meeting,
REALLY IMPORTANT LAB MEETING. SHOW UP. BRING SUMMARIES.
FINAL DAY BEFORE WIKI FREEZES.
Nov. 4, 2008: 7:00 PM Lab Meeting,
REALLY IMPORTANT LAB MEETING. SHOW UP.
LAST LAB MEETING. PRESENTATION PRACTICE AND PREP FOR TRAVEL
Journal Club
8/12/08:Fluorescent Proteins- Ingrid and James
Fluorescent Protein Powerpoint , Paper: Green Fluorescent Protein as a Marker for Gene Expression
8/19/08: Yeast Mating Pathway/MAP Kinase Pathway- Jasper and Tejas
MAP Kinase Powerpoint , Paper: Map Kinase Scaffold
8/26/08: Yeast Promoters- Allison and Nate
Gene structure powerpoint
Paper:
Genomic Footprinting of the Promoter Regions of STE2 and STE3 genes in the Yeast Saccharomyces cerevisiae
Additional Reading:
Interspecies variation reveals a conserved repressor of alpha-specific genes in Saccharomyces yeast
A genomic code for nucleosome positioning
09/02/08: Mating Type Regulation -Brian and Jonathan
Mating Type Regulation Powerpoint:
Papers:
Herskowitz- A Regularory Hierarchy for Cell Specialization in Yeast
09/09/08: Yeast as a model organism- Ambhi and Raghav
Yeast as a model organism (ppt presentation)
Papers:
Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis
The original Yeast two hybrid paper
Data
To upload data, go [http://www.jhu.edu/iGEM/X_files/Read2.html here], click on [http://www.jhu.edu/iGEM/X_files/Read2.html upload data], and provide the necessary information and results.
How to submit data:
1. log-in as you once had to from the www.jhu.edu/iGEM website "login"
- User: ****** etc...
- Pass: ***** etc...
2. click on UPLOAD DATA from the 'x-files page'
3. add data etc.... and click submit: This generates a webpage and the URL to it is linked in the page you are directed to after you press submit. Copy that URL and past it into the wiki or into the web-browser url box to see what it looks like.
\* If you find that the picture you are uploading is not showing up e-mail Tejas.
Alternatively, you can also just upload files directly to the iGEM wiki. Either way is fine.
Status Reports
The status reports of each group below were continuously updated as we worked on the biobricks.
Click on the name of each group to find past status reports throughout the sex detector project.
To learn more about each biobrick, please refer to the Biobrick page.
GROUP 1: Fluorescent Proteins
Summary for Fluorescent Proteins Group
Venus YFP (BBa_K110021) was able to be constructed and sequence verified(with one possible mutation), however due to being unable to RE digest the insert out of the biobrick vector, possibly due to contamination, the part was not sumbittied to the registry... yet. We will submit it after it is verified, after the competition. To see info about this biobrick check out oru patrs: http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Johns_Hopkins
GROUP 2: MATa Specific-promoters
Summary for MATa Specific Promoters Group
We were able to produce sequence-verified clones of BBa_K110008 and BBa_K110016: http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Johns_Hopkins .
GROUP 3: Short Two-Way Stops
We were able to produce a truncated two-way terminator (thus a one way) from the STE2 gene - BBa_K110012. Please see Link for information: http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Johns_Hopkins
GROUP 4: Long Two-Way Stops & MAT(alpha) Specific Promoters
Summary for Long Two-Way Stops & MAT(alpha) Specific Promoters Group
We were able to produce BBa_K110005 and BBa_K110006 (alpha Promoters) in iGEM vectors. Also BBa_K110003 was able to be produced and sequence verified. Check out these parts here http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Johns_Hopkins
GROUP 5: MATa Specific Promoters II
Summary for MATa Specific Promoters II Group
Part Description: Ste2 Promoter Reverse Summary here: We were able to grow colonies with sequence verified Ste2 promoters. The promoter is currently located in a glycerol stock in pGEM vector. One attempt to transfer to the iGEM vector has been made, unsuccessfully. This was due to a mistake in protocol however, so final transfer to the iGEM vector should still be straightforward. Progress on this bio-brick has been paused in order to focus on MFA1, which is more applicable to our project design because this Ste2 designed for the wrong direction. Part no.: BBa_K110015 Part Description: MFA1 Promoter Forward Summary here: We have been unable to get successful, sequence verified clones with our MFA1 promoter thus far. We currently have a few samples of DNA ready to be tested by restriction enzyme digest, and then, hopefully, sequencing.
For more information about the parts go to: http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008& group=Johns_Hopkins
GROUP 6: Vectors
Summary for Vectors Group
Date: July 31, 2008 Status report by: Tejas Part no.: BBa_K1100XX -> BBa_K1100YY Part Description: Vectors for concatenating and executing BioBricks The vectors to be used were delivered to us in STABS from MIT. They are pSB4A5 (amp), pSB4C5 (cam), pSB3K5 (kan), and pSB4K5 (kan). The vectors come pre-inserted with the ccdB gene, preventing growth in all E. coli strains except DB3.1. Total amount of DNA extracted from mini -prep is estimated between 5 to 20 ug per vector. Restriction digests confirmed the extracted DNA was vectors.([http://www.jhu.edu/iGEM/Group6:Vectors/2008-7-24.Vectors%20(CAM,AMP,KAN3,KAN4)%20after%20Digest.Tejas.html Digestion of all four vectors]). Vectors were transformed into JM109, BB#2198 (DB3.1), and BB#5777 (DB3.1). All gave expected results. 1 ug of each vector was digested for future use. Gel of digestion shows DNA is correct. ([http://www.jhu.edu/iGEM/Group6:Vectors/2008-8-12.Digest%20of%20vectors,%20ready%20for%20distribution.Tejas.html Vectors ready for insertions])
GROUP 7: Microscopy/Yeast
Summary for Microscopy/Yeast Group
Date: Oct 27, 2008 Status report by: Tejas Part no.: no Biobrick part Part Description: Yeast vector pRS414 to be temporarily used. The yeast vector pRS414 will be temporarily used until it can be adjusted to meet registry standards. pRS414 contains one EcoRI site and one PstI site, both directly adjacent to each other. This may make digest a little less efficient. Procedure involves first digesting with Pst1 for one hour, then digesting with EcoRI overnight (sequentially, not simultaneously), as EcoRI can tolerate low overhangs better than PstI, according to the NEB catalog. The following is a gel run on an E/P simultaneous digest of pRS414. Lane one is log 2 ladder. Lane 2 is uncut pRS414 (supercoiled so runs fater). Lane 3 is cut vector. It may have only one cut, with half at EcoRI and half at PstI, depending on efficiency of the second cut occuring after the first.
pRS414 digest
GROUP 8: Assembly Progress
Summary of Biobrick Assemblies:
Planned Schedule for Further Work:
October 31st- Successful transformants from either set of ligations (of Promoter + Fluorescent Protein) and transformations will be grown up for Miniprep.
November 1st- These pieces will be miniprepped and digested. Digested fragments will be ligated together with the terminator in the middle, into a pRS414 yeast/bacterial shuttle vector. Non-digested fragments of a promoter + fluorescent protein will also individually be transformed into yeast, with hopes that they will fluoresce.
November 3rd- Full construct insert will be transformed into yeast, pending identity verification.
November 6th- Pretty Yeast! (Hopefully).
