Team:Johns Hopkins/Notebook

http://www.jhu.edu/iGEM/Group6:Vectors/2008-8-12.Digest%20of%20vectors,%20ready%20for%20distribution.Tejas.html

Groups

iGEM Groups 1.0
iGEM Groups 2.01
iGEM Groups 2.02

Important reminders and notes

 [Can 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.
          Be prepared for 20 min Journal club.
 August 12: Tuesday lab meeting. First Journal club topic: fluorescent proteins; James and Ingrid.

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.

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 Biobrick page.

Status Report 2.1 - 07/12/08
Status Report 2.2 - 07/17/08
Status Report 2.3 - 08/07/08

Please BOLD 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.

GROUP 1: Fluorescent Proteins

Summary for Fluorescent Proteins Group

 Date: July 22, 2008
 Status report by: Tejas, Ingrid
 Part no.: BBa_K110017 -> BBa_K110023
 Part Description: yESapphire , mCherry, venusYFP, and Citrine 

 Work on yESapphire was gratiously done by James. Primers were designed. Restriction site ends have been added
 through PCR. The product was cloned into JM109. Colonies were picked and an inoculation was grown. Miniprep
 was performed and subsequent DNA was CS PCR'd and run on a gel to verify contents.
 ([http://www.jhu.edu/iGEM/Group3:ShortTwo-wayStops/2008-7-22.Short%202Way%20Stop,%20Alpha%20Promoters,%20&%20Sapphire%20FP.Allison%20Suarez%20and%20Nate%20Sotuyo%20.html  Short 2Way Stop, Alpha Promoters, & Sapphire FP]) BioBrick is currently being sequenced. 

 Work on mCherry and venusYFP (BBa_K110018 -> BBa_K110021) is currently being done. Primers were designed.
 Restriction sites have been added through PCR.  ([http://www.jhu.edu/iGEM/Group1:FluorescentProteins/2008-7-23.PCR%20Products:%20mCherry%20and%20Venus%20YFP,%20both%20RtL%20and%20LtR.Ingrid,%20Tejas.html PCR Products: mCherry and Venus YFP, both RtL and LtR]) The
 products were cloned into JM109 and plated. 3 colonies per 4 BioBricks (total 12) were picked, grown out,
 mini prepped, and digested for verification on a gel.

 According to the results, ([http://www.jhu.edu/iGEM/Group1:FluorescentProteins/2008-7-22.Venus%20YFP%20and%20mCherry%20Miniprep%20check%20via%20digest.Ingrid.html Venus YFP and mCherry Miniprep check via digest]) , only one of the mCherry's
 (BBa_K110019) is the correct product. A second Digest was preformed to check this, meanwhile new colonies
 have been picked on 7.22.2008 and are being grown out for a higher yield of DNA so that it can be sent off
 for sequencing. [http://www.jhu.edu/iGEM/Group1:FluorescentProteins/2008-7-23.Re-Digest%20of%20three%20samples%20of%20mCherry%20BBa_K110018.Ingrid%20.html Re-Digest of three samples of mCherry BBa_K110018]

 Work on Citrine has not yet begun. Primers have been designed and ordered, but template DNA must be grown out.
 Template DNA will be grown and extracted by James should we later decide that Citrine will be needed.
  
 *Note that mCherry and Citrine both have restriction sites within the coding region, and are therefore not
 optimal. Advice/help on this issue would be appreciated.

GROUP 2: MATa Specific-promoters

Summary for MATa Specific Promoters Group

 Date: August 12, 2008
 Status report by: Allison and Nate
 Part no.: BBa_K11008 -> BBa_K110016
 Part Description: A promoters MFA1 (L+R) and Ste2 (R+L)
 Two more minipreps were completed. DNA concentrations were much higher (>150 ng/ul).

 Date: August 5, 2008
 Status report by: Allison and Nate
 Part no.: BBa_K110008 -> BBa_K110016
 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 will be done to generate more clones with the 
 correct sequence. BBa_K110008 had one mutation; additional minipreps from other clones
 are ready to send off for sequencing, although the DNA concentrations are low. Colonies
 can be picked and overnight cultures grown
 for another round of minipreps.

 Date: July 29, 2008
 Status report by: Allison and Nate
 Part no.: BBa_K110008 -> BBa_K110016
 Part Description: A promoters: MFA1 (L+R) and Ste2 (R+L), respectively.
 Two samples of mini preps from MFA1 and Ste2 were sent off for sequencing at the end of last week.
 Results will follow soon.

GROUP 3: Short Two-Way Stops

Summary for Short Two-Way Stops Group

 Date 8/12/08
 Status report by: Raghav and James
 Part no.:BBa_K110011 -> BBa_K110013; BBa_K110010;17
 Part Description:
 Summary:Short two way stops should be sent out for sequencing this week Needed 
 so much DNA to visualize one band of short two way stop. Sapphire FP progress
 has slowed, due to extraneous bands in gel and extraneous sequence from the trace 
 files. transformations from the biobrick book were tried again with very poor
 results... 1 COLONY... POSSIBLY  THE BIOBRICK BOOK IS UPSETTING.

 Date 8/5/08
 Status report by: James
 Part no.: BBa_K110011 -> BBa_K110013; BBa_K110010;17
 Part Description: Short two way stops; and Sapphire Fluorescent Proteins
 Summary: The sequence data from last week, turned out all right
 both orientations of Sapphire FP should be correct sequences 
 Sequence Data  HOORAY!
 Last weeks miniprep and digestion yielded no DNA, so this week the minprep was repeated and the
 digestion of the prodeuct can be found here RE digest [http://www.jhu.edu/iGEM/Group3:ShortTwo-wayStops/2008-8-5.Two%20way%20stop%20digestion%208/05/08.Jasper;%20James;%20Raghav.html Two way stop digestion 8/05/08]

 Date 7/29/08
 Status report by: James
 Part no.: BBa_K110011 -> BBa_K110013; BBa_K110010;17
 Part Description: Short two way stops
 Summary: Sequencing of BBa_K110010;17 (Sapphire FP) revealed confusing, if not bad
 sequences.Sequence Data  
 PCR cloning and transformation was completed sucessfully last week on the Short two way stops
 A Miniprep was done and a  RE digest will be completed soon (possibly tonight)

 Date: 7/22/2008
 Status report by: James
 Part no.: BBa_K110011 -> BBa_K110013
 Part Description: Short two way stops
 Summary: RE digestion showed no insert the plasmid of our clones, thus the PCR was done again.
 [http://www.jhu.edu/iGEM/Group3:ShortTwo-wayStops/2008-7-23.Short%20way%20stops.James.html Short way stops]

GROUP 4: Long Two-Way Stops & MAT(alpha) Specific Promotors

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

 Date: July 29, 2008
 Status report by: Jaime
 Part no.: BBa_K110001, BBa_K110003, BBa_K110005, BBa_K110006
 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.

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. To produce sufficient quantities of thee vectors, I grew out the strains and performed
 several minipreps. Total amount of DNA 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 Vectors after Digestion with EcoRI and PstI])
 Transformation into DB3.1 competent cells was unsuccessful, likely
 due to the possibility that the competent cells may not actually be DB3.1. Currently making new competent
 cells from old DB3.1 strain (BB #5777) and from new DB3.1 strain (BB #2198).

GROUP 7: Microscopy/Yeast

Summary for Microscopy/Yeast Group

 Date: _________ __, 2008
 Status report by: _____
 Part no.: BBa_K1100XX -> BBa_K1100YY
 Part Description:
 
 Summary here.