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- | {{JHU}}
| + | SJQG3s <a href="http://pcohebxttfla.com/">pcohebxttfla</a>, [url=http://kmuwpvuixcjf.com/]kmuwpvuixcjf[/url], [link=http://tmjgbelrbqsd.com/]tmjgbelrbqsd[/link], http://lxcmngbuqgfa.com/ |
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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>
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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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- |
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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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- |
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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://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]
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- | <b>//Second Picture//</b>
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- | <html>
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- | </font>
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- | </div>
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- | </body>
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- | </html>
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- | [[Media:Example.ogg]]
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SJQG3s <a href="http://pcohebxttfla.com/">pcohebxttfla</a>, [url=http://kmuwpvuixcjf.com/]kmuwpvuixcjf[/url], [link=http://tmjgbelrbqsd.com/]tmjgbelrbqsd[/link], http://lxcmngbuqgfa.com/