Team:Johns Hopkins/Notebook

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.
               BE PREPARAED FOR A LONG NIGHT OF PREPARATION.

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: [[Media:Example.ogg]]
Papers:
[[Media:JHU_0708_paper_Aregularoryheirarchy.pdf|JHU_0708_paper_Aregularoryheirarchy.pdf]]
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.

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

 Date: Oct 19, 2008
 Status report by: Tejas
 Part no.: BBa_K110017 -> BBa_K110023
 Part Description: yESapphire , mCherry, venusYFP, and Citrine 

 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,
 fluoroescent proteins (yeast optimized) sent us as STABS from MIT are showing promise, and may be
 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.

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 Promotors

Summary for Long Two-Way Stops & MAT(alpha) Specific Promoters Group

 Summary: As of September 23rd, 2008
 We had successfully cloned BBa_K110005 and BBa_K110006 (alpha Promoters) and 
 BBa_K110003 (Long Terminator) into pGEM-T and Kanamycin biobrick vectors.  
 They await assembly and ligation.
 For more information on these parts go please visit our sandbox:
 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

 Date: August 12, 2008
 Status report by: Richard Group
 Part no.: BBa_K110009
 Part Description: Ste2
 Summary here: We hit a dead end when we ran out of the miniprep DNA. We are going to have to start
 again from scratch        I suppose.
 Part no.: BBa_K110015
 Part Description: Mfa1
 Summary here: Same place as on Ste2.

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). 

 Date: Week of 10/25/08-11/2/08
 Name: J and J
 Summary:  Using Qiagen Kits, new minipreps and RE digests were completed for:
    Venus YFP (BBa_K110022) 
    alpha promoters (BBa_110005, BBa_K110006)
    a promoter (BBa_K110016)
    mCherry (BBa_E2060)
    dsRed (BBa_E1010)
 See Picture- Media:081026kitEXSPindALL.tif
 Annotations of picture-
[http://www.jhu.edu/iGEM/GroupOther:Other/2008-10-28.Restriction%20Digest%20of%20Team%20Parts.James.html Restriction Digest of Team Parts]

 These fragments were gel purified, and are our current working batch.  They appear to be far 
 cleaner (less contaminating protein and DNA), and digests have produced cut fragments of high 
 enough concentrations to work with (finally).  Assembly ligations of promoter + fluorescent
 protein have been re-attempted, again into pBB414 (CAMr) vectors as well as the pRS414 
 yeast/bacterial shuttle vector.

 Past Miniprep methods have proven difficult (STET BOILING). These digests were cut from
 the gel and ligated together with PBC SK(-) Bacterial plasmid, and transformed into DH5alpha 
 competent cells. The transformation failed, either due to:
 1)	The close proximity of E and P sites at the MCS, so close that each interferes with 
       restriction enzyme binding of the other.
 2)	a mislabeled PBC sK-, lacking chloramphenicol resistance.
       A transformation was set up on the 28th with cut pRS414 (yeast bacteria shuttle
       vector), as well as another   attempted ligation with PBC SK(-). pUC19 was also cut 
       and ligations will be attempted on the 29th. 

 Date: October 22-25, 2008
 Authors: James/Jonathan
 We have made repeated attempts to digest our minipreps with restriction enzymes for assmebly {E/S 
 or X/P) but all gels produce either not enough cut product to work with or the digest runs extremely
 messily.  
 One example: //picture//

 The reasons for this are most likely twofold:
   a)  The miniprep method (STET Boiling lysozyme protocol) does not remove a lot of contaminating
       proteins and unwanted DNA, producing very 'dirty' DNA.
   b)  The proper balance between restriction enzyme and miniprep DNA amounts has not yet been reached.
       Too much DNA may inhibit enzyme digestion, and may account for a lack of clear banding at the 
       expected product size range.
 We will continue to adjust the protocol to attempt a clean cut.

 Date: Week of October 20th, 2008
 Authors: James/Jonathan
 Several things- 
 All initial biobricks were digested with E/P to confirm their identity-where possible, 
 bacterial perms in pGEM-T were used, due to biobrick vectors not producing high-copy numbers.  
 The digest photographs can be found here:
 //pic//
 All digests ran far too slow, possibly from overloading of enzyme.  Gels were very messy, and 
 there are plans to repeat using proper concentrations of miniprep DNA vs. restriction enzymes.

 Simultaneously, PCR was performed on all biobrick parts again.  If in pGEM, T7/SP6 
 promoters were used, and parts in biobrick vectors were amplified using the iGEM sequencing 
 primers.  See: //PICTURE//

 The PCR was directly taken and digested with proper enzymes for assembly (E/S or X/P), gel 
 purified, and then ligated together into a vector pBB414 (Cam resistant).  Unfortunately, 
 the Taq polymerase was not disabled, and most likely polymerized at the restriction overhangs, 
 leading to poor transformation counts.

 Date: October 12th, 2008
 Author: Jonathan
 PCR was performed on all assembly minipreps using the iGEM vR and F primers 
 (normally used for sequencing).  All lanes yielded unusual results, none of 
 which are at the expected product size.
 PCR Gel picture:
 //picture//
 *Note: iGEM primers are each approximately 150 bp away from cloning site, 
 so products will be +300 bp than expected

 Date: October 3rd, 2008
 Author: Jonathan
 All assemblies were digested with enzyme Nsp1 - the normal Chloramphenicol biobrick 
 vector (without no insert)has Nsp1 sites approximately opposite each other, so digestion
 should yield a fragment of one size, half the normal Cam vector size.
 If there is an insert present, digestion will yield two bands of different sizes.
 Results of Nsp1 digest:
 //Picture//
 While BBa_K110005 has an Nsp1 site within its sequence, the fragmentation pattern 
 of the bands is still bizarre.

 Date: October 2nd, 2008
 Author: Jonathan
 Preps or previous clones were re-made, using a Qiagen kit and column.  Quantities were too 
 low to work with (<40ng/ul)

 Date:  Week of September 22nd, 2008
 Author: Jonathan
 Performed the following digest on all available biobrick parts, to begin assembly process:
 
 Part:              Orientation:   BBPart#/Clone:  Vector:    Enzymes Used:
 Alpha Promoter     LtR            BBa_K110005/1   pGEM-T     E/S
 Alpha Promoter     LtR            BBa_K110006/1   pGEM-T     E/S
 a Promoter         RtL            BBa_K110016/1   Cam BBV    X/P
 mCherry FP         LtR            BBa_E2050       Kan BBV    X/P
 mCherry FP         LtR            BBa_E2060       Kan BBV    X/P
 dsRed FP           LtR            BBa_E1010       Kan BBV    X/P
 Venus YFP          LtR            BBa_K110020/1   pGEM-T     X/P
 Venus YFP          RtL            BBa_K110021/2   pGEM-T     E/S
 Long Terminator     -             BBa_K110003/2   pGEM-T     E/P

 Each promoter was ligated to a flourescent protein of the proper orientation (i.e. Alpha Promoter 
 5 to mCherry 2050, 2060, and dsRed 1010 and Venus 20, etc.):

 Alpha Promoter 5.1 + mCherry 2050           
 Alpha Promoter 5.1 + mCherry 2060
 Alpha Promoter 5.1 + dsRed1010
 Alpha Promoter 6.1 + mCherry 2050
 Alpha Promoter 6.1 + mCherry 2060
 Alpha Promoter 6.1 + dsRed1010
 Alpha Promoter 5.1 + Venus 20.1
 Alpha Promoter 6.1 + Venus 20.1
 a Promoter 16.1 + Venus YFP 21.1
 
 Each was assembled into the chloramphenicol biobrick vector, ligated O/N, 
 The next day, each was transformed into DH5alpha cells (along with a -insert and negative control), 
 and then plated on LB cam plates.  12 colonies were picked for each ligation, and 4 of each assmebly
 was miniprepped.  
 Digestion by E/P yielded  (Gel has two rows):
 [http://www.jhu.edu/iGEM/GroupOther:Other/2008-10-28.9/25/08%20Assemblies%20-%20Upper%20Row.Jonathan%20&%20James.html 9/25/08 Assemblies - Upper Row]
 //Second Picture//