Team:Johns Hopkins/Notebook/GROUP 8: Assembly Progress

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 assembly 
 {E/S or X/P) but all gels produce either not enough cut product to work with or the digest 
 runs extremely  messily. See: [http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=16&rid=4991 Individual Part Assembly Enzyme Digestion, 2 and 5ul]
 This particular digest produced some purifiable bands for the promoters, but overall not enough 
 digested product could be extrated.

 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:
 [http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=16&rid=4993 Initial Digest Verification- Messy]
 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: [http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=16&rid=4992 Biobrick Part PCR]
 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:
 [http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=16&rid=4973 PCR of all BBV parts and assemblies]
 *Note: iGEM primers are each approximately 150 bp away from cloning site, 
 so products will be +300 bp than expected.  Lower row is another Nsp1 digest- it failed, but
 that was 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:
 [http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=16&rid=4990 Nsp1 Digest Results]
 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://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=16&rid=4988 Upper Row]
 [http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=16&rid=4989 Lower Row]