Team:Johns Hopkins/Notebook

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Latest revision as of 04:50, 30 October 2008

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
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).

@@ Line 1: / Line 1: @@
 {{JHU}}
-== Files ==
+== Groups ==
 [[Media:JHU_0708_IGEMgroups.pdf|iGEM Groups 1.0]]<br>
 [[Media:JHU_iGEM_groups2.01.pdf|iGEM Groups 2.01]]<br>
@@ Line 6: / Line 6: @@
 == Important reminders and notes ==
-   [Can make general comments here, so they don't get lost in peoples e-mail boxes]<br>
+   [Make general comments here, so they don't get lost in peoples e-mail boxes]
-   [For anyone not part of the iGEM team who would like to see the gels we're running:
-. Click the links provided
+   July 11: Primers for group 1 were delivered yesterday.
-. Guest login: bluejays <br>
+   July 11: Lab meeting at 7:30PM in the lab to go over miniprep protocol.
-   July 11:
+   July 15: Lab meeting at 6:30PM with Jessica. Have status reports ready.
-  Primers for group 1 were delivered yesterday<br>
+           Bring labtop if you can.
-   July 11:
+   July 17: Restriction Digest/Sequencing Preparation (with James) 6:00PM.
-  Lab meeting at 7:30PM in the lab to go over miniprep protocol<br>
+   July 21: 6:00PM or 6:30PM Lab meeting with Jessica. Have status reports ready.
-   Tuesday July 15:
+  July 29: 7:00PM Lab Meeting. Meet in Conference Room across from lab. Have Status Reports ready
-  Lab meeting at 6:30PM with Jessica. Have status reports ready.
+  Aug. 05: 7:00PM Lab Meeting. Meet in Conference Room across from lab. Have Status Reports ready.
-  Bring labtop if you can <br>
+  Aug. 12: 7:00PM Lab Meeting. Have Status Reports Ready.
-   July 17:
+                Journal Club Topic: Fluorescent Proteins; James and Ingrid.
-  Restriction Digest/Sequencing Preparation (with James) 6:00PM<br>
+  Aug. 19: 7:00PM Lab Meeting. Have Status Reports Ready.
-   July 21:
+                Journal Club Topic: Yeast Mating Pathway/MAP Kinase Pathway ; Jasper and Tejas
-:00PM or 6:30PM Lab meeting with Jessica. Have status reports ready.
+  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,
+                <b>REALLY IMPORTANT LAB MEETING. SHOW UP. BRING SUMMARIES.
+                FINAL DAY BEFORE WIKI FREEZES.</b>
+  Nov. 4, 2008: 7:00 PM Lab Meeting,
+                <b>REALLY IMPORTANT LAB MEETING. SHOW UP.
+                LAST LAB MEETING. PRESENTATION PRACTICE AND PREP FOR TRAVEL</b>
-== Status Reports ==
+== Journal Club ==
+<html>
+/12/08:<b>Fluorescent Proteins</b>- Ingrid and James<br>
+</html>
+[[Media:JHU_Fluorescent_Protein_Journal_Club.ppt| Fluorescent Protein Powerpoint]] <br> Paper: [[Media:Green_Fluorescent_Protein_as_a_Marker_for_Gene_Expression.pdf|Green Fluorescent Protein as a Marker for Gene Expression]]<br>
+<html><br>
-The status reports of each group below will continuously be updated as we work on the biobricks. The following PDFs contain progressive versions of our status reports as we continue through the sex detector project; they are added weekly. To learn more about each biobrick, please refer to the [[Team:Johns_Hopkins/Biobricks|Biobrick]] page.
+/19/08: <b>Yeast Mating Pathway/MAP Kinase Pathway-</b> Jasper and Tejas<br>
+</html>
+[[Media:MAPK_Pathway.ppt | MAP Kinase Powerpoint]] , Paper: [[Media:MapK_Scaffold_SynBio_Paper.pdf | Map Kinase Scaffold]]<br>
+<html><br>
-[[media:JHU_0708_Status_reports2.1.pdf|Status Report 2.1]] - 07/12/08 <br>
+/26/08: <b>Yeast Promoters</b>- Allison and Nate<br>
-[[media:JHU_0708_Status_reports2.2.pdf‎|Status Report 2.2]] - 07/17/08 <br>
+</html>
+[[Media:Gene_sturucture_powerpoint.ppt| Gene structure powerpoint]]<br>
+Paper:<br>[[Media:Genomic_Footprinting_of_the_Promoter_Regions_of_STE2_and_STE3_genes_in_the_Yeast_Saccharomyces_cerevisiae.pdf| Genomic Footprinting of the Promoter Regions of STE2 and STE3 genes in the Yeast Saccharomyces cerevisiae]]<br>
+Additional Reading: <br>[[Media:JHU_0808_Interspeciesvariation.pdf‎|Interspecies variation reveals a conserved repressor of alpha-specific genes in Saccharomyces yeast]]<br>
+[[Media:JHU_0808_Agenomiccode.pdf|A genomic code for nucleosome positioning]]
+<html><br>
+<br>
-Please<b> BOLD </b>the most recent step that you have completed. Do this by placing the tag < b > in front of and </ b > at the end of what you would like to be placed in bold (with no space between letter and carrot symbol (<,>).
+/02/08: <b> Mating Type Regulation </b>-Brian and Jonathan <br>
+Papers:  <br></html>
+[[Media:JHU_0708_paper_Aregularoryheirarchy.pdf|Herskowitz- A Regularory Hierarchy for Cell Specialization in Yeast]]<br>
+<html>
+<br>
+/09/08: <b>Yeast as a model organism</b>- Ambhi and Raghav<br>
+</html>
+[[Media:Model organism.ppt| Yeast as a model organism (ppt presentation)]]<br>
+Papers:<br>
+[[Media:Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.pdf| Functional characterization of the ''S. cerevisiae'' genome by gene deletion and parallel analysis ]]<br>
+[[Media:A novel genetic system to study protein protein interactions.pdf‎|The original Yeast two hybrid paper]]<br>
+<html><br>
-=== GROUP 1: Fluorescent Proteins ===
+</html>
-  Date: July 1 2008
+== Data ==
-  status report by: Ingrid (work done by James)
-  Part no.: BBa_K110017
-  Part Description: yESapphire
-  Part Location (in build a genome lab): In James and Jasper's PCR product Box,
-  Stainless Steel 4 degree
-  PCR successful?; Yes
-  Cloning of PCR product successful: Y/N
-  Sequencing of cloned PCR product successful: Not done
-  Joining of validated part to adjacent part(s) status: Not done
-  Current status of this part: This part is being Sequenced
-  Date: July 1 2008
+To upload data, go <b>[http://www.jhu.edu/iGEM/X_files/Read2.html here]</b>, click on <b>[http://www.jhu.edu/iGEM/X_files/Read2.html upload data]</b>, and provide the necessary information and results.
-  status report by: Ingrid (work done by James)
-  Part no.: BBa_K110010
-  Part Description: yESapphire
-  Part Location (in build a genome lab): In James and Jasper's PCR product Box,
-  Stainless Steel 4 degree
-  PCR successful?; Yes
-  Cloning of PCR product successful: Y/N
-  Sequencing of cloned PCR product successful: No
-  Joining of validated part to adjacent part(s) status: Not done
-  Current status of this part: This part is being sequenced
-  Date: July 17 2008
+<b><h5>How to submit data:</h5></b>
-  status report by: Ingrid
+. log-in as you once had to from the www.jhu.edu/iGEM website "login"
-  Part no.: BBa_K110018
+*User: ****** etc...
-  Part Description: mCherry:
+*Pass: ***** etc...<br>
-  Part Location (in build a genome lab): In silver fridge by door
+. click on UPLOAD DATA from the 'x-files page'<br>
-  PCR successful?; Yes
+. 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.<br>
-  (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1471)
+\* If you find that the picture you are uploading is not showing up e-mail Tejas.
-  <b>Cloning of PCR product successful: Inconclusive
-  http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1476</b>
-  Sequencing of cloned PCR product successful: Not done
-  Joining of validated part to adjacent part(s) status: Not done
-  Problems to be solved: Had some extra unknown products, with unknown bands in the gel
-  Current status of this part: Done with touchdown PCR and gel. Next step is to clone product.
-  Date: July 17 2008
+Alternatively, you can also just upload files directly to the iGEM wiki. Either way is fine.
-  status report by: Ingrid
-  Part no.: BBa_K110019
-  Part Description: mCherry:
-  Part Location (in build a genome lab): In silver fridge by door
-  PCR successful?; Yes
-  (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1471)
-  <b>Cloning of PCR product successful: Inconclusive
-  http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1476</b>
-  Sequencing of cloned PCR product successful: No
-  Joining of validated part to adjacent part(s) status: Not done
-  Problems to be solved: Need to figure out if gel after digest shows correct products
-  Date: July 17 2008
+== Status Reports ==
-  status report by: Ingrid
-  Part no.: BBa_K110020
-  Part Description: Venus Enhanced YFP
-  Part Location (in build a genome lab): In silver fridge by door
-  PCR successful?; Yes
-  (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1471)
-  <b>Cloning of PCR product successful: inconclusive
-  http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1476</b>
-  Sequencing of cloned PCR product successful: No
-  Joining of validated part to adjacent part(s) status: Not done
-  Problems to be solved: Had some extra unknown products, with unknown bands in the gel
-  Current status of this part: Done with touchdown PCR and gel. Next step is to clone product.
-  Date: July 17 2008
-  status report by: Ingrid
-  Part no.: BBa_K110021
-  Part Description: Venus Enhanced YFP
-  Part Location (in build a genome lab): In silver fridge by door
-  PCR successful?; Yes
-  (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1471)
-  <b>Cloning of PCR product successful: Inconclusive
-  http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1476</b>
-  Sequencing of cloned PCR product successful: No
-  Joining of validated part to adjacent part(s) status: Not done
-  Problems to be solved: Had some extra unknown products, with unknown bands in the gel
-  Current status of this part: Done with touchdown PCR and gel. Next step is to clone product.
-=== GROUP 2: MATa Specific-promoters ===
-  Status report by Allison and Nate
+The status reports of each group below were continuously updated as we worked on the biobricks.<br>
-  Part no.: BBa_K110008
+<b>Click on the name of each group to find past status reports throughout the sex detector project.</b>
-  Part Description: MFA1 (L+R)
-  Part Location: in a labeled box, second shelf from the top, -20
-  degrees C refrigerator next to front door
-  Date: 7/10/08
-  PCR successful? Yes
-  http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&advanced=0&paging=&page=33
-  Cloning of PCR product successful: in progress
-  Sequencing of cloned PCR product successful: not done
-  Joining of validated part to adjacent part(s) status: not done
-  Problems to be solved: to be determined
-  Current status of this part: PCR was being troubleshooted, appeared to
-  have good results with regular PCR protocol (not touchdown) in which
-  there was a constant annealing temperature of 55 degrees C - see gel
-  Status report by Allison and Nate
+To learn more about each biobrick, please refer to the [[Team:Johns_Hopkins/Biobricks|Biobrick]] page.
-  Part no.: BBa_K110016
-  Part Description: Ste2 (R+L)
-  Part Location: in a labeled box, second shelf from the top, -20
-  degrees C refrigerator next to front door
-  Date: 7/10/08
-  PCR successful? Yes
-  http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&advanced=0&paging=&page=33
-  Cloning of PCR product successful: in progress
-  Sequencing of cloned PCR product successful: not done
-  Joining of validated part to adjacent part(s) status: not done
-  Problems to be solved: to be determined
-  Current status of this part: Both PCR protocols (touchdown and second
-  PCR with constant annealing temperature) produced product of the
-  correct size. BBa_K110016 was used as a control in the second PCR with
-  BBa_K110008.
-  Status report by Allison and Nate
-  Part no.: BBa_K110008
-  Part Description: MFA1 (L+R)
-  Part Location: same as above, plates are at 4 degrees refrigerator near front door
-  Date: 7/14/08
-  PCR successful? Yes
-  Cloning of PCR product of successful? There were mainly light blue colonies
-   (only a couple white colonies)
-  Sequencing of cloned PCR product successful: not done
-  Joining of validated part to adjacent part(s) status: not done
-  Problems to be solved: Ligation
-  Current status of this part: plates are at 4 degrees; another ligation/transformation
-   will be completed soon
-  Status report by Allison and Nate
-  Part no.: BBa_K110016
-  Part Description: Ste2 (R+L)
-  Part Location: in a labeled box, second shelf from the top, -20 degrees C refrigerator next to
-   front door; plates at 4 degrees
-  Date: 7/14/08
-  PCR successful? Yes
-  Cloning of PCR product successful: There were many blue colonies (similar to the plate of BB_K110008)
-  Sequencing of cloned PCR product successful: not done
-  Joining of validated part to adjacent part(s) status: not done
-  Problems to be solved: Ligation
-  Current status of this part: plates are at 4 degrees; another ligation/transformation
-   will be completed soon
-  Status report by Allison and Nate
+=== [[Team:Johns Hopkins/Notebook/GROUP 1: Fluorescent Proteins | GROUP 1: Fluorescent Proteins]] ===
-  Part no.: BBa_K110008
-  Part Description: MFA1 (L+R)
-  Part Location: same as above, plates are at 4 degrees refrigerator near front door
-  Date: 7/22/08
-  PCR successful? Yes (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&mode=single&page=2
-  Cloning of PCR product of successful? Yes (approx 60 white colonies between two plates)
-  Sequencing of cloned PCR product successful: not done
-  Joining of validated part to adjacent part(s) status: not done
-  Problems to be solved:
-  Current status of this part: 12 mini preps and the restriction digestion were completed;
-  preparing to send 3 samples to be sequenced
-  (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1478)
-  Status report by Allison and Nate
+	<b>Summary for Fluorescent Proteins Group</b>
-  Part no.: BBa_K110016
-  Part Description: Ste2 (R+L)
-  Part Location: in a labeled box, second shelf from the top, -20 degrees C refrigerator next to
-   front door; plates at 4 degrees
-  Date: 7/22/08
-  PCR successful? Yes
-  Cloning of PCR product successful: Yes (approx 20 white colonies on one plate)
-  Joining of validated part to adjacent part(s) status: not done
-  Problems to be solved:
-  Current status of this part: 12 mini preps and the restriction digestion were completed;
-  preparing to send 3 samples to be sequenced
-  (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1478)
-=== GROUP 3: Short two way stops ===
+  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
-  Date: 7/21/08
+=== [[Team:Johns Hopkins/Notebook/GROUP 2: MATa Specific-promoters | GROUP 2: MATa Specific-promoters]] ===
-  status report by: James
-  Part no.: BBa_K110011
-  Part Description: Between-bud 27-W FRS2-C LtR
-  Part Location (in build a genome lab): In James and Jasper's PCR product Box,
-   Stainless Steel 4 degree
-  PCR successful?; Yes
-  Cloning of PCR product successful: Yes
-  (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1475)
-  Sequencing of cloned PCR product successful: No
-  Joining of validated part to adjacent part(s) status: Not done
-  Problems to be solved:
-  Current status of this part: Miniprep digestion yielded no product, PCR; ligation;
-  transformation; and miniprep will be repeated by this Wednesday (07/23/08)
-  status report by: James
+<b>Summary for MATa Specific Promoters Group</b>
-  Part no.: BBa_K110012
-   Part Description: Between STE2-W and BST1-C LtR
+   We were able to produce sequence-verified clones of BBa_K110008 and BBa_K110016:
-  Part Location (in build a genome lab): In James and Jasper's PCR product Box,
+   http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Johns_Hopkins .
-   Stainless Steel 4 degree
-  PCR successful?; Yes
-  Cloning of PCR product successful: Yes
-   (http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1475)
-  Sequencing of cloned PCR product successful: No
-  Joining of validated part to adjacent part(s) status: Not done
-  Problems to be solved:
-  Current status of this part: Miniprep digestion yielded no product, PCR; ligation;
-  transformation; and miniprep will be repeated by this Wednesday (07/23/08)
-  status report by: James
+=== [[Team:Johns Hopkins/Notebook/GROUP 3: Short two way stops | GROUP 3: Short Two-Way Stops]] ===
-  Part no.: BBa_K110013
-  Part Description: Between-SWP82-W and EMP47-C LtR
-  Part Location (in build a genome lab): In James and Jasper's PCR product Box,
-   Stainless Steel 4 degree
-  PCR successful?; No
-  Cloning of PCR product successful: No
-  Sequencing of cloned PCR product successful: No
-  Joining of validated part to adjacent part(s) status: Not done
-  Problems to be solved: The PCR of this part yielded a very large product
-  Current status of this part:
-=== GROUP 4: Long Two-way Stops & Mat(alpha) specific promotors ===
+  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
+=== [[Team:Johns Hopkins/Notebook/GROUP 4: Long Two-way Stops & Mat(alpha) specific promoters | GROUP 4: Long Two-Way Stops & MAT(alpha) Specific Promoters]] ===
-Date: 7/10/08
+<b>Summary for Long Two-Way Stops & MAT(alpha) Specific Promoters Group</b>
-  Status report by: Jaime Liu
-  Part no.: BBa_K110001
-  Part Description:
-  BBa_K110001 - Between-bud 27-W FRS2-C + 200bp into each gene LtR
-  Part Location (in build a genome lab): In 4C fridge #2
-  PCR successful?; Y/N (link such as this)- Yes
-  BBa_K110001: http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1462
-  Cloning of PCR product successful: Y/N  Yes
-  Sequencing of cloned PCR product successful: Y/N  No
-  Joining of validated part to adjacent part(s) status:  Not Done
-  Problems to be solved: Not really a problem, but need do a mini-Prep and sequence
-  Current status of this part: All cloned and inoculated into 100uL in 96 well plate for
-  sequencing- put in incubator on 7/15
-Date: 7/10/08
+   We were able to produce BBa_K110005 and BBa_K110006 (alpha Promoters) in iGEM vectors.
-   Status report by: Jaime Liu
+   Also BBa_K110003 was able to be produced and sequence verified. Check out these parts here
-  Part no.: BBa_K110003
+   http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Johns_Hopkins
-  Part Description:
-   BBa_K110003 - Between-SWP82-W and EMP47-C +200 into each gene LtR
-   Part Location (in build a genome lab): In 4C fridge #2
-  PCR successful?; Y/N (link such as this)- Yes
-  BBa_K110003: http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1463
-  Cloning of PCR product successful: Y/N  Yes
-  Sequencing of cloned PCR product successful: Y/N  No
-  Joining of validated part to adjacent part(s) status:  Not Done
-  Problems to be solved: Not really a problem, but need do a mini-Prep and sequence
-  Current status of this part: All cloned and inoculated into 100uL in 96 well plate for
-  sequencing- put in incubator on 7/15
-Date: 7/10/08
+=== [[Team:Johns Hopkins/Notebook/GROUP 5: MATa Specific Promoters II | GROUP 5: MATa Specific Promoters II]] ===
-  Status report by: Jaime Liu
-  Part no.: BBa_K110005
-  Part Description:
-  BBa_K110005 - MFalpha2 LtR
-  Part Location (in build a genome lab): In 4C fridge #2
-  PCR successful?; Y/N (link such as this)- Yes
-  BBa_K110005: http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1470
-  Cloning of PCR product successful: Y/N  Yes
-  Sequencing of cloned PCR product successful: Y/N  No
-  Joining of validated part to adjacent part(s) status:  Not Done
-  Problems to be solved: Not really a problem, but need do a mini-Prep and sequence
-  Current status of this part: All cloned and inoculated into 100uL in 96 well plate for
-  sequencing- put in incubator on 7/15
-Date: 7/10/08
+<b>Summary for MATa Specific Promoters II Group</b>
-  Status report by: Jaime Liu
+   Part Description: Ste2 Promoter Reverse
-   Part no.: BBa_K110006
+   Summary here: We were able to grow colonies with sequence verified Ste2 promoters.
-   Part Description:
+    The promoter is currently located in a glycerol stock in pGEM vector. One attempt to
-  BBa_K110006 - MFalpha1 LtR
+    transfer to the iGEM vector has been made, unsuccessfully. This was due to a mistake
-  Part Location (in build a genome lab): In 4C fridge #2
+    in protocol however, so final transfer to the iGEM vector should still be straightforward.
-  PCR successful?; Y/N (link such as this)- Yes
+    Progress on this bio-brick has been paused in order to focus on MFA1, which is more
-  BBa_K110006: http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=4&rid=1470
+    applicable to our project design because this Ste2 designed for the wrong direction.
-   Cloning of PCR product successful: Y/N  Yes
-   Sequencing of cloned PCR product successful: Y/N  No
+   Part no.: BBa_K110015
-   Joining of validated part to adjacent part(s) status:  Not Done
+   Part Description: MFA1 Promoter Forward
-   Problems to be solved: Not really a problem, but need do a mini-Prep and sequence
+   Summary here: We have been unable to get successful, sequence verified clones with
-  Current status of this part: All cloned and inoculated into 100uL in 96 well plate for
+    our MFA1 promoter thus far. We currently have a few samples of DNA ready to be tested
-  sequencing- put in incubator on 7/15
+    by restriction enzyme digest, and then, hopefully, sequencing.
-=== GROUP 5: MATa Specific Promoters II ===
+ For more information about the parts go to:
+ http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Johns_Hopkins
-  Date: 7/11/08
+=== [[Team:Johns Hopkins/Notebook/GROUP 6: Vectors | GROUP 6: Vectors]] ===
-  status report by Rick Carrick
-  Part no.: BBa_K110015,
-  Part Description: MFA1
-  Part Location (in build a genome lab):
-  PCR successful?; Y (on moodle somewhere)
-  Cloning of PCR product successful: Y
-  Sequencing of cloned PCR product successful:N
-  Joining of validated part to adjacent part(s) status: Not done
-  Problems to be solved: None so far
-  Current status of this part: This parts must be restriction enzyme digested and sequenced
-  next.
-  Date: 7/11/08
+<b>Summary for Vectors Group</b>
-  status report by Rick Carrick
-  Part no.: BBa_K110009
-  Part Description: Ste2
-  Part Location (in build a genome lab):
-  PCR successful?; Y (on moodle somewhere)
-  Cloning of PCR product successful: Y
-  Sequencing of cloned PCR product successful:N
-  Joining of validated part to adjacent part(s) status: Not done
-  Problems to be solved: None so far
-  Current status of this part: This part must be restriction enzyme digested and sequenced <br> next
-=== GROUP 6: Vectors ===
+  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.<b>([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])</b>.
+  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.
+  <b>([http://www.jhu.edu/iGEM/Group6:Vectors/2008-8-12.Digest%20of%20vectors,%20ready%20for%20distribution.Tejas.html Vectors ready for insertions])</b>
-  Status report by ____
+=== [[Team:Johns Hopkins/Notebook/GROUP 7: Microscopy/Yeast | GROUP 7: Microscopy/Yeast]] ===
-  Vector transformed into bacteria strain DB3.1 Y/N
-  Permanent culture made in the Boeke lab for future reference Y/N
-  Selectable marker for this vector
-  Medium made and tested Y/N  (link)
-  DNA preps made Y/N
-  DNA preps tested by RE digest  - (link)
-  DNA preps tested by transformation into DB3.1 and DH5alpha (or JM109) – put data as
-    Table on moodle.
-  Sample format/data follows:<br>
-  Amount transformed		cfu/micGm in DB3.1		cfu/micGm in JM109
-.1 ng					5 * 10e7			<2 * 10e2<br>
-  Preparative digests ready for use are located – where?
-=== GROUP 7: Microscopy/Yeast ===
+<b>Summary for Microscopy/Yeast Group</b>
-   Milestones
+   Date: Oct 27, 2008
-   Have gfp yeast been visualized by microscopy?
+   Status report by: Tejas
-   Have gfp yeast been photographed? Link to moodle – picture.
+   Part no.: no Biobrick part
-   [If pics are nice, put on our web page]
+  Part Description: Yeast vector pRS414 to be temporarily used.
-   Can green(/other colors) colonies be photographed?
-   Has STE3-gfp sex detector been transformed into MATa, MATalpha, and MATa/alpha?  Y/N
+  The yeast vector pRS414 will be temporarily used until it can be adjusted to meet registry
-   Have permanent cultures been banked in the Boeke lab? Y/N
+  standards. pRS414 contains one EcoRI site and one PstI site, both directly adjacent to each
-   Have STE3-gfp sex detector cells/colonies been photographed Y/N  - link to Moodle
+   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.<br>
+   [[Media: PRS414.tif|pRS414 digest]]
+=== [[Team:Johns Hopkins/Notebook/GROUP 8: Assembly Progress | GROUP 8: Assembly Progress]] ===
+<b>Summary of Biobrick Assemblies:</b>
+  Planned Schedule for Further Work:<br>
+  October 31st- Successful transformants from either set of ligations (of Promoter + Fluorescent
+  Protein) and transformations will be grown up for Miniprep. <br>
+  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.<br>
+  November 3rd- Full construct insert will be transformed into yeast, pending identity verification.<br>
+  November 6th- Pretty Yeast! (Hopefully). <br>
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