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

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Planned Schedule for Further Work:<br>
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  October 31st- Successful transformants from either set of ligations (of Promoter + Fluorescent
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  Protein) and transformations will be grown up for Miniprep. <br>
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  November 1st- These pieces will be miniprepped and digested. Digested fragments will be ligated
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  together with the terminator in the middle, into a pRS414 yeast/bacterial shuttle vector. 
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  Non-digested fragments of a promoter + fluorescent protein will also individually be transformed
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  into yeast, with hopes that they will fluoresce.<br>
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  November 3rd- Full construct insert will be transformed into yeast, pending identity verification.<br>
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  November 6th- Pretty Yeast! (Hopefully). <br>
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  Date: Week of 10/25/08-11/2/08
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  Name: J and J
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  Summary:  Using Qiagen Kits, new minipreps and RE digests were completed for:
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    Venus YFP (BBa_K110022)
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    alpha promoters (BBa_110005, BBa_K110006)
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    a promoter (BBa_K110016)
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    mCherry (BBa_E2060)
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    dsRed (BBa_E1010)
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  See Picture- [[Media:081026kitEXSPindALL.tif]]
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  Annotations of picture-
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[http://www.jhu.edu/iGEM/GroupOther:Other/2008-10-28.Restriction%20Digest%20of%20Team%20Parts.James.html Restriction Digest of Team Parts]<br>
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  These fragments were gel purified, and are our current working batch.  They appear to be far
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  cleaner (less contaminating protein and DNA), and digests have produced cut fragments of high
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  enough concentrations to work with (finally).  Assembly ligations of promoter + fluorescent
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  protein have been re-attempted, again into pBB414 (CAMr) vectors as well as the pRS414
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  yeast/bacterial shuttle vector.<br>
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  Past Miniprep methods have proven difficult (STET BOILING). These digests were cut from
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  the gel and ligated together with PBC SK(-) Bacterial plasmid, and transformed into DH5alpha
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  competent cells. The transformation failed, either due to:
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  1) The close proximity of E and P sites at the MCS, so close that each interferes with
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        restriction enzyme binding of the other.
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  2) a mislabeled PBC sK-, lacking chloramphenicol resistance.
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        A transformation was set up on the 28th with cut pRS414 (yeast bacteria shuttle
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        vector), as well as another  attempted ligation with PBC SK(-). pUC19 was also cut
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        and ligations will be attempted on the 29th.
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  Date: October 22-25, 2008
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  Authors: James/Jonathan
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  We have made repeated attempts to digest our minipreps with restriction enzymes for assmebly {E/S
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  or X/P) but all gels produce either not enough cut product to work with or the digest runs extremely
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  messily. 
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  One example: <b>//picture//</b><br> abab
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  The reasons for this are most likely twofold:
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    a)  The miniprep method (STET Boiling lysozyme protocol) does not remove a lot of contaminating
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        proteins and unwanted DNA, producing very 'dirty' DNA.
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    b)  The proper balance between restriction enzyme and miniprep DNA amounts has not yet been reached.
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        Too much DNA may inhibit enzyme digestion, and may account for a lack of clear banding at the
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        expected product size range.
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  We will continue to adjust the protocol to attempt a clean cut.
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  Date: Week of October 20th, 2008
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  Authors: James/Jonathan
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  Several things-
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  All initial biobricks were digested with E/P to confirm their identity-where possible,
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  bacterial perms in pGEM-T were used, due to biobrick vectors not producing high-copy numbers. 
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  The digest photographs can be found here:
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  <b>//pic//</b>
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  All digests ran far too slow, possibly from overloading of enzyme.  Gels were very messy, and
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  there are plans to repeat using proper concentrations of miniprep DNA vs. restriction enzymes.<br>
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  Simultaneously, PCR was performed on all biobrick parts again.  If in pGEM, T7/SP6
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  promoters were used, and parts in biobrick vectors were amplified using the iGEM sequencing
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  primers.  See: <b>//PICTURE//</b><br>
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  The PCR was directly taken and digested with proper enzymes for assembly (E/S or X/P), gel
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  purified, and then ligated together into a vector pBB414 (Cam resistant). Unfortunately,
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  the Taq polymerase was not disabled, and most likely polymerized at the restriction overhangs,
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  leading to poor transformation counts.
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  Date: October 12th, 2008
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  Author: Jonathan
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  PCR was performed on all assembly minipreps using the iGEM vR and F primers
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  (normally used for sequencing).  All lanes yielded unusual results, none of
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  which are at the expected product size.
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  PCR Gel picture:
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  <b>//picture//</b>
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  *Note: iGEM primers are each approximately 150 bp away from cloning site,  
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  so products will be +300 bp than expected
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  Date: October 3rd, 2008
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  Author: Jonathan
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  All assemblies were digested with enzyme Nsp1 - the normal Chloramphenicol biobrick
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  vector (without no insert)has Nsp1 sites approximately opposite each other, so digestion
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  should yield a fragment of one size, half the normal Cam vector size.
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  If there is an insert present, digestion will yield two bands of different sizes.
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  Results of Nsp1 digest:
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  <b>//Picture//</b>
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  While BBa_K110005 has an Nsp1 site within its sequence, the fragmentation pattern
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  of the bands is still bizarre.
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  Date: October 2nd, 2008
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  Author: Jonathan
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  Preps or previous clones were re-made, using a Qiagen kit and column.  Quantities were too
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  low to work with (<40ng/ul)
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  Date:  Week of September 22nd, 2008
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  Author: Jonathan
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  Performed the following digest on all available biobrick parts, to begin assembly process:
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  Part:              Orientation:  BBPart#/Clone:  Vector:    Enzymes Used:
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  Alpha Promoter    LtR            BBa_K110005/1  pGEM-T    E/S
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  Alpha Promoter    LtR            BBa_K110006/1  pGEM-T    E/S
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  a Promoter        RtL            BBa_K110016/1  Cam BBV    X/P
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  mCherry FP        LtR            BBa_E2050      Kan BBV    X/P
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  mCherry FP        LtR            BBa_E2060      Kan BBV    X/P
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  dsRed FP          LtR            BBa_E1010      Kan BBV    X/P
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  Venus YFP          LtR            BBa_K110020/1  pGEM-T    X/P
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  Venus YFP          RtL            BBa_K110021/2  pGEM-T    E/S
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  Long Terminator    -            BBa_K110003/2  pGEM-T    E/P<br>
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  Each promoter was ligated to a flourescent protein of the proper orientation (i.e. Alpha Promoter
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  5 to mCherry 2050, 2060, and dsRed 1010 and Venus 20, etc.):<br>
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  Alpha Promoter 5.1 + mCherry 2050         
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  Alpha Promoter 5.1 + mCherry 2060
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  Alpha Promoter 5.1 + dsRed1010
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  Alpha Promoter 6.1 + mCherry 2050
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  Alpha Promoter 6.1 + mCherry 2060
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  Alpha Promoter 6.1 + dsRed1010
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  Alpha Promoter 5.1 + Venus 20.1
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  Alpha Promoter 6.1 + Venus 20.1
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  a Promoter 16.1 + Venus YFP 21.1
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  Each was assembled into the chloramphenicol biobrick vector, ligated O/N,
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  The next day, each was transformed into DH5alpha cells (along with a -insert and negative control),
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  and then plated on LB cam plates.  12 colonies were picked for each ligation, and 4 of each assmebly
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  was miniprepped. 
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  Digestion by E/P yielded  (Gel has two rows):
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  [http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=16&rid=4988 Upper Row]
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  [http://baderlab.bme.jhu.edu:8888/moodle18/mod/data/view.php?d=16&rid=4989 Lower Row]
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[[Media:Example.ogg]]
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Latest revision as of 15:57, 28 March 2016

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