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 31: / Line 31: @@
    Oct. 28, 2008: 7:00 PM Lab Meeting,
                  <b>REALLY IMPORTANT LAB MEETING. SHOW UP. BRING SUMMARIES.
-                 BE PREPARAED FOR A LONG NIGHT OF PREPARATION.</b>
+                 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>
 == Journal Club ==
@@ Line 37: / Line 41: @@
 /12/08:<b>Fluorescent Proteins</b>- Ingrid and James<br>
 </html>
-[[Media:JHU_Fluorescent_Protein_Journal_Club.ppt| Fluorescent Protein Powerpoint]] , Paper: [[Media:Green_Fluorescent_Protein_as_a_Marker_for_Gene_Expression.pdf|Green Fluorescent Protein as a Marker for Gene Expression]]<br>
+[[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>
@@ Line 54: / Line 58: @@
 <br>
+/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>
+[[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>
@@ Line 80: / Line 89: @@
 == Status Reports ==
-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.
+The status reports of each group below were continuously updated as we worked on the biobricks.<br>
+<b>Click on the name of each group to find past status reports throughout the sex detector project.</b>
+To learn more about each biobrick, please refer to the [[Team:Johns_Hopkins/Biobricks|Biobrick]] page.
-[[media:JHU_0708_Status_reports2.1.pdf|Status Report 2.1]] - 07/12/08 <br>
-[[media:JHU_0708_Status_reports2.2.pdf‎|Status Report 2.2]] - 07/17/08 <br>
-[[media:080708JHU_StatusReport2.3.pdf|Status Report 2.3]] - 08/07/08 <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 (<,>). Click on your group name for the detailed changes occurring to each biobrick.
 === [[Team:Johns Hopkins/Notebook/GROUP 1: Fluorescent Proteins | GROUP 1: Fluorescent Proteins]] ===
@@ Line 92: / Line 100: @@
 	<b>Summary for Fluorescent Proteins Group</b>
-   Date: Oct 19, 2008
+   Venus YFP (BBa_K110021) was able to be constructed and sequence verified(with one possible
-  Status report by: Tejas
+   mutation), however due to being unable to RE digest the insert out of the biobrick
-  Part no.: BBa_K110017 -> BBa_K110023
+   vector, possibly due to contamination,  the part was not sumbittied to the registry... yet.
-  Part Description: yESapphire , mCherry, venusYFP, and Citrine <br>
+   We will submit it after it is verified, after the competition.
-   Work on YFP has progressed, though the biobrick system is giving diffiulty in obtaining a useful
-   construct. Moreover, the YFP sequence may not fully match the expected sequence. However,
+   To see info about this biobrick check out oru patrs:
-   fluoroescent proteins (yeast optimized) sent us as STABS from MIT are showing promise, and may be
+  http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Johns_Hopkins
-  ready for some use by the Jamboree date if all goes well. Work will continue on venusYFP and
-   mCherry (though mCherry unfortunately contains a restriction site). Sapphire must be started over
-   sue to technical difficulties getting a clone.
 === [[Team:Johns Hopkins/Notebook/GROUP 2: MATa Specific-promoters | GROUP 2: MATa Specific-promoters]] ===
 <b>Summary for MATa Specific Promoters Group</b>
-  Date: August 19, 2008
-   Status report by: Allison and Nate
+   We were able to produce sequence-verified clones of BBa_K110008 and BBa_K110016:
-  Part no.: BBa_K110008 -> BBa_K110016
+   http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Johns_Hopkins .
-   Part Description: A promoters: MFA1 (L+R) and Ste2 (R+L), respectively.
-  Sequences were analyzed. BBa_K110016 had a perfect clone. Since there is not much miniprep left,
-  a transformation was done to generate more clones with the correct sequence. BBa_K110008 had one
-  mutation. Another transformation into a Kan BioBrick vector was attempted but was unsuccessful.
-  September 2: Transformation into a Cam BioBrick was completed, and the restriction digestion of
+=== [[Team:Johns Hopkins/Notebook/GROUP 3: Short two way stops | GROUP 3: Short Two-Way Stops]] ===
-  the Biobricks from the pGEM vector were checked, comparing the inserts intended to be ligated to
-  the Kan Biobrick vector and the inserts intended to be ligated to the Cam Biobrick vector;
-  notice that the old inserts are either too faint to see or are not there:
-  [[Media:lanes2thru5OldDigest0816NewDigest0816.jpg|lanes2thru5OldDigest0816NewDigest0816]]
-  Lanes: 1 (2-log ladder), 2 BBa_K110008 old, 3 BBa_K110016 old, 4 BBa_K110008 new, 5 BBa_K110016
-  new, 6 (2-log ladder)
-  A restriction digestion with EcoRI and PstI was done to check for the insert after ligated and
-  transformed with the Cam Biobrick vector: [[Media:090308digest08_16cam.jpg|090308digest08_16_cam]]
-  Lanes: 1 (old aliquot of 2-log ladder), 2 (new aliquot of 2-log ladder), 3 skip, 4 BBa_K110008,
-skip, 6 BBa_K110016
-   September 7: Since the restriction digestions have been unclear as to whether or not the ligation
+   We were able to produce a truncated two-way terminator (thus a one way)
-   of MFA1 and STE2 into the Cam BioBrick vector was successful, another transformation was done
+   from the STE2 gene - BBa_K110012. Please see Link for information:
-  into the Cam Biobrick vector, restriction digestions on the previous mini prep from the 1st
+   http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Johns_Hopkins
-  transformation and 2nd transformation completed, as well as PCR on the mini preps using the
-  corresponding primers for BBa_K11008 and BBa_K110016. In the gel: after the 2 log ladder, the
-  next for lanes have the restriction digestion products (approx 500 ng of plasmid each) and the
-  second four lanes are pcr products in the following order: 08 (1st cam transformation) 16 (1st
-   cam transformation) 08 (2nd transformation) 16 (2nd transformation) for both the restriction
-  digestion and the pcr. The final lane was a control used in the pcr, unrelated to the project.
-  [[Media:090708digestpcr0816.jpg|090708digestpcr0816]]
-  The restriction digestion did not show the insert, while pcr showed a band of the expected size
-  in two of the four samples.
-=== [[Team:Johns Hopkins/Notebook/GROUP 3: Short two way stops | GROUP 3: Short Two-Way Stops]] ===
+=== [[Team:Johns Hopkins/Notebook/GROUP 4: Long Two-way Stops & Mat(alpha) specific promoters | GROUP 4: Long Two-Way Stops & MAT(alpha) Specific Promoters]] ===
-=== [[Team:Johns Hopkins/Notebook/GROUP 4: Long Two-way Stops & Mat(alpha) specific promotors | GROUP 4: Long Two-Way Stops & MAT(alpha) Specific Promotors]] ===
 <b>Summary for Long Two-Way Stops & MAT(alpha) Specific Promoters Group</b>
-   Date: July 29, 2008
+   We were able to produce BBa_K110005 and BBa_K110006 (alpha Promoters) in iGEM vectors.
-  Status report by: Jaime
+  Also BBa_K110003 was able to be produced and sequence verified. Check out these parts here
-  Part no.: BBa_K110001, BBa_K110003, BBa_K110005, BBa_K110006
+   http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Johns_Hopkins
-  Part Description: Long Two-way Stops & Mat(alpha) specific promotors
-   [http://www.jhu.edu/iGEM/Group4:LongTwo-wayStopsANDMATalphaSpecificPromoters/2008-7-25.Restriction%20Enzyme%20Digest%20of%20Mini-Preps.Jaime.html Restriction Enzyme Digest of Mini-Preps]
-  Summary here:
-  Need to be sequenced.
 === [[Team:Johns Hopkins/Notebook/GROUP 5: MATa Specific Promoters II | GROUP 5: MATa Specific Promoters II]] ===
 <b>Summary for MATa Specific Promoters II Group</b>
+   Part Description: Ste2 Promoter Reverse
-  Date: August 12, 2008
+   Summary here: We were able to grow colonies with sequence verified Ste2 promoters.
-  Status report by: Richard Group
+    The promoter is currently located in a glycerol stock in pGEM vector. One attempt to
-  Part no.: BBa_K110009
+    transfer to the iGEM vector has been made, unsuccessfully. This was due to a mistake
-   Part Description: Ste2
+    in protocol however, so final transfer to the iGEM vector should still be straightforward.
-   Summary here: We hit a dead end when we ran out of the miniprep DNA. We are going to have to start
+    Progress on this bio-brick has been paused in order to focus on MFA1, which is more
-  again from scratch        I suppose.
+    applicable to our project design because this Ste2 designed for the wrong direction.
    Part no.: BBa_K110015
-   Part Description: Mfa1
+   Part Description: MFA1 Promoter Forward
-   Summary here: Same place as on Ste2.
+   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
 === [[Team:Johns Hopkins/Notebook/GROUP 6: Vectors | GROUP 6: Vectors]] ===
@@ Line 195: / Line 176: @@
    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
+   The yeast vector pRS414 will be temporarily used until it can be adjusted to meet registry
-  site and one PstI site, both directly adjacent to each other. This may make digest a little less efficient. Procedure involves first
+  standards. pRS414 contains one EcoRI site and one PstI site, both directly adjacent to each
-  digesting with Pst1 for one hour, then digesting with EcoRI overnight (sequentially, not simultaneously), as EcoRI can tolerate low
+  other. This may make digest a little less efficient. Procedure involves first digesting with
-  overhangs better than PstI, according to the NEB catalog. The following is a gel run on an E/P simultaneous digest of pRS414.
+  Pst1 for one hour, then digesting with EcoRI overnight (sequentially, not simultaneously), as
-   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
+  EcoRI can tolerate low overhangs better than PstI, according to the NEB catalog. The following
-  half at EcoRI and half at PstI, depending on efficiency of the second cut occuring after the first.<br>
+  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]]
@@ Line 206: / Line 190: @@
 <b>Summary of Biobrick Assemblies:</b>
-   Date:
+   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>
-[http://www.jhu.edu/iGEM/GroupOther:Other/2008-10-28..James.html ]
 <html>
    </font>