iGEM 2008 — Team Johns_Hopkins

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# Team:Johns Hopkins/Notebook

### From 2008.igem.org

|  |
| --- |
| Contents   * [1 Groups](#Groups) * [2 Important reminders and notes](#Important_reminders_and_notes) * [3 Journal Club](#Journal_Club) * [4 Data](#Data)   + [4.1 How to submit data:](#How_to_submit_data:) * [5 Status Reports](#Status_Reports)   + [5.1 GROUP 1: Fluorescent Proteins](#GROUP_1:_Fluorescent_Proteins)   + [5.2 GROUP 2: MATa Specific-promoters](#GROUP_2:_MATa_Specific-promoters)   + [5.3 GROUP 3: Short Two-Way Stops](#GROUP_3:_Short_Two-Way_Stops)   + [5.4 GROUP 4: Long Two-Way Stops & MAT(alpha) Specific Promoters](#GROUP_4:_Long_Two-Way_Stops_.26_MAT.28alpha.29_Specific_Promoters)   + [5.5 GROUP 5: MATa Specific Promoters II](#GROUP_5:_MATa_Specific_Promoters_II)   + [5.6 GROUP 6: Vectors](#GROUP_6:_Vectors)   + [5.7 GROUP 7: Microscopy/Yeast](#GROUP_7:_Microscopy.2FYeast)   + [5.8 GROUP 8: Assembly Progress](#GROUP_8:_Assembly_Progress) |

## Groups

[iGEM Groups 1.0](/wiki/images/3/34/JHU_0708_IGEMgroups.pdf "JHU 0708 IGEMgroups.pdf")
[iGEM Groups 2.01](/wiki/images/f/f8/JHU_iGEM_groups2.01.pdf "JHU iGEM groups2.01.pdf")
[iGEM Groups 2.02](/wiki/images/3/3a/JHU_iGEM_groups2.02.pdf "JHU iGEM groups2.02.pdf")

## 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](/wiki/images/b/bf/JHU_Fluorescent_Protein_Journal_Club.ppt "JHU Fluorescent Protein Journal Club.ppt")

## 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'

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](/Team%3AJohns_Hopkins/Biobricks "Team:Johns Hopkins/Biobricks") page.

### [GROUP 1: Fluorescent Proteins](/Team%3AJohns_Hopkins/Notebook/GROUP_1%3A_Fluorescent_Proteins "Team:Johns Hopkins/Notebook/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](/Team%3AJohns_Hopkins/Notebook/GROUP_2%3A_MATa_Specific-promoters "Team:Johns Hopkins/Notebook/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](/Team%3AJohns_Hopkins/Notebook/GROUP_3%3A_Short_two_way_stops "Team:Johns Hopkins/Notebook/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](/Team%3AJohns_Hopkins/Notebook/GROUP_4%3A_Long_Two-way_Stops_%26_Mat%28alpha%29_specific_promoters "Team:Johns Hopkins/Notebook/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](/Team%3AJohns_Hopkins/Notebook/GROUP_5%3A_MATa_Specific_Promoters_II "Team:Johns Hopkins/Notebook/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](/Team%3AJohns_Hopkins/Notebook/GROUP_6%3A_Vectors "Team:Johns Hopkins/Notebook/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](/Team%3AJohns_Hopkins/Notebook/GROUP_7%3A_Microscopy/Yeast "Team:Johns Hopkins/Notebook/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](/Team%3AJohns_Hopkins/Notebook/GROUP_8%3A_Assembly_Progress "Team:Johns Hopkins/Notebook/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).
```