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==Important Stuff that was done (in reverse chronological order)==
==About Me==
===7/31/07===
I am currently a graduate student in the joint program between [http://www.fas.harvard.edu/~biophys/ Harvard Biophysics] and [http://hst.mit.edu/index.jsp HST].
====Colony PCR of luxpR Round 2 Take 1====
Colony PCR'ed the transformation plates from last night. The transformation plates looked very good, with lots of colonies.


[[IGEM:Harvard/2007/GeorgeXu/070731ColonyPCRgx|Gel]]
==Research History==
===2005 Summer===
I studied [http://cesonoma.ucdavis.edu/files/45907.pdf Berry Shrivel Disease] under the mentorship of [http://sandtiger.dbs.ucdavis.edu/FacultyProfiles/PlantBiologyGG/DisplayFacultyProfile.cfm?ResearcherID=1426 Professor Ken Shackel] and [http://matthews.ucdavis.edu/BS.html Dr. Mark Krasnow] at UC Davis's Department of Viticulture and Enology through the [http://ysp.ucdavis.edu/ Young Scholar's Program]. I imaged grape berry sections bathed in [http://en.wikipedia.org/wiki/Fluorescein_diacetate_hydrolysis fluorescein diacetate], which is intracellularly hydrolyzed by viable cells to the fluorescent protein fluorescein <cite> FDA </cite>, in order to assess cell viability in diseased grapes. Our hypothesis was that the disease was caused by massive cell death, in particular the [http://en.wikipedia.org/wiki/Companion_cells#Companion_cells companion cells] surrounding the phloem. I had to leave for school before distinct symptoms presented and my data could be analyzed, but later analysis proved inconclusive. I can no longer find the CD with all of my 1000+ awesome images, but you can see a sample on slide 23 of [http://matthews.ucdavis.edu/Power%20Point%20Presentations/ASEV.ppt Dr. Krasnow's presentation].


===7/30/07===
===2007 Spring===
====luxpR Construction Round 2====
During the second semester of my freshman year, I briefly worked part-time in the [http://www.physics.harvard.edu/people/facpages/weitz.html Weitz Lab] with [http://people.seas.harvard.edu/~rowat/ Dr. Amy Rowat] on a microfluidics device for fluorescent detection, actuation, and control of individual yeast cells for directed evolution studies.
Miniprep, Digest, Dephosphorylate, Clonewell, Ligate, Transform:
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0062 R0062]<[http://parts.mit.edu/registry/index.php/Part:BBa_A340620 A340620]
*[http://parts.mit.edu/registry/index.php/Part:BBa_C0061 C0061]<[http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]


The only difference is that the Minipreps turned out a lot better, giving nanodrop values of >130 ng/µL. Also, I stupidly transformed into Top10F' instead of Top10.
===2007 Summer - 2007 Winter===
During the summer following my freshman year I was a member of [[IGEM:Harvard/2007|Harvard's 2007 iGEM team]]. The overall goal of our project was to have bacteria that would bind to a target and activate a signal, either through quorum sensing or transmembrane signal transduction. Our project consisted of three modules:
# We planned to utilize surface expression of a peptide library through fusion with the surface proteins Lpp-OmpA <cite> LppOmpA </cite> and autodisplay protein AIDA-1 <cite> AIDA1 </cite> for screening of binding sequences. We were able demonstrate successful fusion and targeting with his and strep2 tags but did not have time to test a random library.
# The project that I helped [[IGEM:Harvard/2007/Brainstorming#Quorum_Sensing|brainstorm]] was the quorum sensing. We utilized [http://www.che.caltech.edu/groups/fha/quorum.html LuxI/LuxR quorum sensing] found in ''V. fishcheri'' to create two systems: one was a single-cell that created both the "sender" LuxI molecule and the "receiver" luxR molecule, while the other had the "sender" and "receiver" constructs in separate cells. We successfully demonstrated quorum sensing in our system and began [http://parts.mit.edu/igem07/index.php/Harvard#Quorum_Sensing|initial tests of combining these constructs with surface expression]. My work on this project is (mostly) documented in my [[User:GeorgeXu/Notebook/iGEM_2007|iGEM Lab Notebook]].
# We planned to construct transmembrane signal transduction using the Fec system <cite>Fec</cite>. Unfortunately, the project turned out to be too ambitious and we were unsuccessful.
Professor George Church was one of the faculty advisors for this program and one of his students, Harris Wang, was one of the Teaching Fellows. After the iGEM jamboree, I began working for Harris in the Church lab.


===7/29/07===
===2008 Summer===
====Colony PCR of luxpR Round 1 Take 2====
During the summer following my sophomore year I received generous funding from the [http://www.fas.harvard.edu/~rijs/fellowships/undergrad.html Harvard Reischauer Institute's Summer Internship Program] to work in Japan for 10 weeks. While there, I prepared and published a review article under the mentorship of Masakazu Umezawa and [http://www.researchsea.com/html/experts.php/eid/379/research/ken_takeda.html Professor Ken Takeda] on the permanent health effects of adverse fetal environments.
I colony PCR'ed the colonies from the transformation plates from last night. The plates were a little better, with around 10 colonies each. I only got *i think* the [http://parts.mit.edu/registry/index.php/Part:BBa_C0061 C0061]<[http://parts.mit.edu/registry/index.php/Part:BBa_I13504 I13504] to work. Also, the five bands that should be [http://parts.mit.edu/registry/index.php/Part:BBa_R0062 R0062]<[http://parts.mit.edu/registry/index.php/Part:BBa_A340620 A340620] are all about the size of [http://parts.mit.edu/registry/index.php/Part:BBa_R0062 R0062] so I'm going to redo the construction tomorrow.


[[IGEM:Harvard/2007/GeorgeXu/070730ColonyPCRgx|Gel]]
===2008 Spring - 2010 Spring===
I worked in the Church lab with Harris Wang and Dr. Farren Isaacs on lambda-red mediated recombineering <cite> lambdared </cite>. Most of my work is documented on an internal lab wiki.


===7/28/07===
==Publications==
====Colony PCR of luxpR Round 1 Take 1====
# Wang HH, Isaacs FJ, Carr PA, Sun ZZ, '''Xu G''', Forest CR, and Church GM. 'Programming cells by multiplex genome engineering and accelerated evolution'. Nature. 460, 894 (2009)
Unfortunately, there were like no colonies. Meaning:
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0062 R0062]<[http://parts.mit.edu/registry/index.php/Part:BBa_A340620 A340620]
**Two colonies
*[http://parts.mit.edu/registry/index.php/Part:BBa_C0061 C0061]<[http://parts.mit.edu/registry/index.php/Part:BBa_I13504 I13504]
**Zero colonies
*[http://parts.mit.edu/registry/index.php/Part:BBa_C0061 C0061]<[http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]
**Two colonies
So I colony PCR'ed them and then froze the result (no gel running today, folks).
===Religating and Transforming===
I re-ligated and transformed some leftover DNA from last night:


*[http://parts.mit.edu/registry/index.php/Part:BBa_R0062 R0062]<[http://parts.mit.edu/registry/index.php/Part:BBa_A340620 A340620]
==References==
*[http://parts.mit.edu/registry/index.php/Part:BBa_C0061 C0061]<[http://parts.mit.edu/registry/index.php/Part:BBa_I13504 I13504]
<biblio>  
*[http://parts.mit.edu/registry/index.php/Part:BBa_C0061 C0061]<[http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]
#FDA pmid=4192549
 
#LppOmpA pmid=11036660
Finally, I transformed the constructs into Top10 cells.
#AIDA1 pmid=9006035
 
#Fec pmid=10931321
===7/27/07===
#lambdared pmid=18265390
====Clonewell, Vacufuge, Ligate, Transform====
#chemotaxis pmid=911982
I clonewelled, vacufuged, and then ligated the following:
</biblio>
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0062 R0062]<[http://parts.mit.edu/registry/index.php/Part:BBa_A340620 A340620]
*[http://parts.mit.edu/registry/index.php/Part:BBa_C0061 C0061]<[http://parts.mit.edu/registry/index.php/Part:BBa_I13504 I13504]
*[http://parts.mit.edu/registry/index.php/Part:BBa_C0061 C0061]<[http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]
 
Finally, I transformed the constructs into BL21 cells.
 
====Dephosphorylating the Vectors====
I dephosphorylated the [http://parts.mit.edu/registry/index.php/Part:BBa_R0062 R0062] and [http://parts.mit.edu/registry/index.php/Part:BBa_C0061 C0061] with Antarctic Phosphotase and then heat inactivated them for 10m at 65dC.
====Digesting for Double luxpR====
I digested the following for 99:99 at 37dC:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''DNA'''
| align="center" style="background:#f0f0f0;"|'''Enzyme 1'''
| align="center" style="background:#f0f0f0;"|'''Enyzme 2'''
| align="center" style="background:#f0f0f0;"|'''Buffer 2'''
| align="center" style="background:#f0f0f0;"|'''BSA'''
|-
| R0062||20 µL||1 µL SpeI||1 µL PstI||2.5 µL||0.5 µL
|-
| A340620||20 µL||1 µL XbaI||1 µL PstI||2.5 µL||0.5 µL
|-
| C0261||20 µL||1 µL SpeI||1 µL PstI||2.5 µL||0.5 µL
|-
| E0240||20 µL||1 µL XbaI||1 µL PstI||2.5 µL||0.5 µL
|-
| I13504||20 µL||1 µL XbaI||1 µL PstI||2.5 µL||0.5 µL
|}
 
====Sequencing the C0261====
I prepared and sent [http://parts.mit.edu/registry/index.php/Part:BBa_C0261 C0261] for sequencing.
 
====Miniprep for Double luxpR====
Perry and I miniprepped:
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0062 R0062]
*[http://parts.mit.edu/registry/index.php/Part:BBa_A340620 A340620]
*[http://parts.mit.edu/registry/index.php/Part:BBa_C0261 C0621]
*[http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]
*[http://parts.mit.edu/registry/index.php/Part:BBa_I13504 I13504]
in order to build the luxpR construct.
 
===7/26/07===
====FACS====
Perry and I diluted down overnight samples and grew them in an incushaker from 9:30-12:30. The numbers indicate OD right before we resuspended in PBS. We tried to resuspend so that the final OD was around 0.003.
*JT
**1:10 (.555)
**1:100 (.141)
**1:1000 (.02)
**1:10000 (.000-.001)
*T02&S23I07 1:1000 (.03)
*T02&J23039 1:1000 (.03)
*F20I07&S23E40 1:1000 (.075)
*J04450 1:100 (.064)
*I13522 1:100 (.62)
*B0015 1:100 (.061)
 
[[Media:072707facsgx+pt.zip]]
 
====Overnight Plate Reader Growth Curve Results====
I set up a plate reader as follows. The numbers after the label indicate starting OD. Note: Our plate reader seems to have trouble reading OD's around 0.1, so I'm not completely sure if the 0.1 values are accurate. I took the lower OD readings in the OD machine in the small room, so I'm pretty confident about those.
 
{| {{table}}
| ||1||2||3||4||5||6||7||8||9||10||11||12
|-
| A||T02<br>0.106||T02<br>0.106||T02<br>0.106||J39<br>0.106||J39<br>0.106||J39<br>0.106||S23I07<br>0.106||S23I07 <br>.106||S23I07<br>0.106||S23E40<br>0.105||S23E40<br>0.105||S23E40<br>0.105
|-
| B||S08E40<br>.109||S08E40<br>.109||S08E40<br>.109||S08I07<br>0.11||S08I07<br>0.11||S08I07<br>0.11||BLANK LB||BLANK LB||BLANK LB||BLANK LB||BLANK LB||BLANK LB
|-
| C||T02<br>0.002||T02<br>0.002||T02<br>0.002||J39<br>0.001||J39<br>0.001||J39<br>0.001||S23I07<br>0.002||S23I07 <br>0.002||S23I07<br>0.002||S23E40<br>0.002||S23E40<br>0.002||S23E40<br>0.002
|-
| D||S08E40<br>.003||S08E40<br>.003||S08E40<br>.003||S08I07<br>0.004||S08I07<br>0.004||S08I07<br>0.004||BLANK LB||BLANK LB||BLANK LB||BLANK LB||BLANK LB||BLANK LB
|-
| E||T02<br>0.043||T02<br>0.043||T02<br>0.043||J39<br>0.038||J39<br>0.038||J39<br>0.038||S23I07<br>0.040||S23I07 0.040||S23I07<br>0.040||S23E40<br>0.041||S23E40<br>0.041||S23E40<br>0.041
|-
| F||S08E40<br>.042||S08E40<br>.042||S08E40<br>.042||S08I07<br>0.045||S08I07<br>0.045||S08I07<br>0.045||BLANK LB||BLANK LB||BLANK LB||BLANK LB||BLANK LB||BLANK LB
|}
 
Results:
* Starting at OD~0.1 or OD~0.04 it took the samples around 2 1/2 hrs to read OD~0.3 and around a total of 6 hrs to hit stationary phase
*Starting at OD~0.003, it took the samples around 4 hours to reach OD~0.3 and around a total of 7 hours to hit stationary phase
* There didn't seem to be a big difference between starting at OD~0.1 vs OD~0.04. Of course, this could be due to the fact that I took the OD reading for the OD~0.1 samples in the plate reader, which is inaccurate in that range.
* Overall, the samples grew at pretty much the same rate. There appears to be at most a 45-60 min lag between different constructs, but this could be due to variations in starting OD.
* The blank LB samples were interesting. They were pretty much flat and low until around 8 hrs in, then they grew exponentially to around OD~1. This seems to indicate that there were very low levels of contamination in these LB wells. I added LB+amp to these wells, so the contamination is probably coming from our own constructs. I don't know if this contamination was present when I loaded the wells or somehow there was cross-well contamination during the reading period.
 
For all the graphs+raw data: [[Media:072607platereadergx.xls]]
 
===7/25/07===
====Sorting through Sequences====
I went through all the sequences we have done and compiled the results
 
[[User:GeorgeXu/Sequences|Sequencing Results]]
 
====Miniprepping, Nanodrop, and Sequencing S03608.I13507====
I miniprepped and sent [http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608]>[http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507] for sequencing. Elapsed time: 20 minutes.
 
====Colony PCR of various parts====
Perry and I colony PCR'ed the following parts:
*[http://parts.mit.edu/registry/index.php/Part:BBa_F2620 F2620] >[http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]
*[http://parts.mit.edu/registry/index.php/Part:BBa_J37015 J37015]
*[http://parts.mit.edu/registry/index.php/Part:BBa_J37034 J37034]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0062 R0062]
*[http://parts.mit.edu/registry/index.php/Part:BBa_I13504 I13504]
*[http://parts.mit.edu/registry/index.php/Part:BBa_J37032 J37032]
*[http://parts.mit.edu/registry/index.php/Part:BBa_A340620 A340620]
*his[http://parts.mit.edu/registry/index.php/Part:BBa_E0040 E0040]mek
 
[[IGEM:Harvard/2007/GeorgeXu/070725ColonyPCRgx|Gel]]
 
===7/24/07===
====Liquid Cultures====
Perry and I grew liquid cultures of several things:
*[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002]
*[http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039]
*[http://parts.mit.edu/registry/index.php/Part:BBa_S03623 S03623] >[http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]
*[http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608] >[http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]
*[http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03623] >[http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]
*[http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608] >[http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]
====Mike's Agar Plate Experiment====
Perry and I carried out a preliminary test of Mike's Plate Reader Experiment.
Protocol:
# Pipet 200 µL of overnight cultures of [http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002] and spread onto LB agar plates.
# Let the plates sit at room temperature to allow the bacteria to soak in. (We let it sit for about 20 minutes)
#Pipet in 10 µL of one of each of the following into the middle of each plate.
## Overnight culture of [http://parts.mit.edu/registry/index.php/Part:BBa_S03623 S03623] > [http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]
## Overnight culture of [http://parts.mit.edu/registry/index.php/Part:BBa_S03623 S03623] > [http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]
## Overnight culture of [http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608] > [http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]
## Overnight culture of [http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608] > [http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]
## 2 µM OHHL
We did duplicates because we took from two different overnight cultures, one that was obviously red and one that was not obviously red.
 
====Ligating and Transforming the RP-JT and FluorSwitch====
Perry and I performed the following ligations and transformations:
*[http://parts.mit.edu/registry/index.php/Part:BBa_F2620 F2620] >[http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]
into BL21
 
We decided not to do the other ligations and transformations because we found out that we already have a [http://parts.mit.edu/registry/index.php/Part:BBa_S03623 S03623] <[http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240] and we were colony PCR'ing a  [http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608]<[http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]. Finally, because the  [http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039] >[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002] miniprep and clonewell sucked, we were reluctant to move forward on that ligation.
 
 
====Clonewell for RP-JT and FluorSwitch====
I clonewelled and vacufuged all the [[User:GeorgeXu#Digest_for_RP-JT_and_FluorSwitch|digests]] I did earlier in the day. The band for [http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039] >[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002] was almost invisible.
 
====Sequencing the F2620 and E0240====
I prepared a sequencing order for [http://parts.mit.edu/registry/index.php/Part:BBa_F2620 F2620] and [http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240].
 
====Dephosphorylate for RP-JT and FluorSwitch====
I dephosphorylated the following:
*[http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039] >[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002]
*[http://parts.mit.edu/registry/index.php/Part:BBa_S03623 S03623]
*[http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608]
*[http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]
with Antarctic Phosphatase for 1 hr at 37dC and then heat inactivated for 10m at 65dC.
 
====Digest for RP-JT and FluorSwitch====
I digested the following for 99m99s.
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''DNA'''
| align="center" style="background:#f0f0f0;"|'''Enzyme 1'''
| align="center" style="background:#f0f0f0;"|'''Enzyme 2'''
| align="center" style="background:#f0f0f0;"|'''Buffer 2'''
| align="center" style="background:#f0f0f0;"|'''BSA'''
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]||21 µL ||1 µL EcoRI||1 µL XbaI||2.5 µL ||0.5 µL
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]||21 µL ||1 µL EcoRI||1 µL SpeI||2.5 µL ||0.5 µL
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039] >[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002]||21 µL ||1 µL EcoRI||1 µL SpeI||2.5 µL ||0.5 µL
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_S03623 S03623]||21 µL ||1 µL XbaI||1 µL PstI||2.5 µL ||0.5 µL
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608]||21 µL ||1 µL XbaI||1 µL PstI||2.5 µL ||0.5 µL
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]||21 µL ||1 µL SpeI||1 µL PstI||2.5 µL ||0.5 µL
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_F2620 F2620]||21 µL ||1 µL EcoRI||1 µL SpeI||2.5 µL ||0.5 µL
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]||21 µL ||1 µL EcoRI||1 µL XbaI||2.5 µL ||0.5 µL
|}
 
====Miniprep (RPJT+FluorSwitch)====
I miniprepped the samples that were grown last night:
* For RP-JT constructions
**[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]
**[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]
**[http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039] >[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002]
* For switching fluorophores
**[http://parts.mit.edu/registry/index.php/Part:BBa_S03623 S03623]
**[http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608]
**[http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]
**[http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]
All the nanodrop data was good (about 150 ng/µL with excellent curves), except for the [http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039] >[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002] (50ng/µL)
 
===7/23/07===
====Colony PCR of RP====
I colony PCR'ed the R-P constructs that didn't work out the first time:
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340]
 
[[IGEM:Harvard/2007/GeorgeXu/070723ColonyPCRgx|Gel]]
 
====Growing Liquid Cultures====
I grew liquid cultures:
* For RP-JT constructions
**[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]
**[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]
**[http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039] >[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002]
* For switching fluorophores
**[http://parts.mit.edu/registry/index.php/Part:BBa_S03623 S03623]
**[http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608]
**[http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]
**[http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]
 
===7/22/07===
====Colony PCR of R-PJT====
[[IGEM:Harvard/2007/GeorgeXu/070722ColonyPCRgx|Gel]]
 
===7/21/07===
====Clonewell RP and JT====
I clonewelled the following things (some are Perry's samples):
*1+2B XP
*JsLO ES (very faint)
*JsLO SP (very faint)
*[http://parts.mit.edu/registry/index.php/Part:BBa_B0015 B0015] EX
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140] colony #1
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #2
 
and then vacufuged them.
 
====Dephosphorylating JT====
I dephosphorylated JT with Antarctic Phosphotase (1 hr 37dC) and then heat inactivated at 65dC for 10 min.
===7/20/07===
====Clonewell and Vacufuging the RP and JT parts====
I clonewelled the leftovers from the RP and JT digests and then vacufuged and resuspended in 20 µL of nuclease free water. This was done in order to try rebuilding the constitutive tetR+JT construct.
 
===7/19/07===
====Liquid Cultures of 2 cell system and RFP+JT====
I grew liquid cultures of the 2 cell system and the RFP+JT in preparation for plate reader experiments tomorrow.
 
====Sequencing the RFP+JT part====
I prepared the RFP+JT part and sent it for sequencing.
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Label'''
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Primer'''
|-
| AV01||J04450-JT||VF2
|-
| AV02||J04450-JT||VR
|-
| AV03||jSLOPDZ2B||VF2
|-
| AV04||jSLOPDZ2B||VR
|-
| AV05||jSLOPDZ1B||VF2
|-
| AV06||jSLOPDZ1B||VR
|-
| AV07||JhisGFPmek||VF2
|-
| AV08||JhisGFPmek||VR
|}
 
====Colony PCR of the tetR+JT constructs Part II====
I chose only non-fluorescent colonies (by checking the colonies under the fluorescent microscope) for this colony PCR.
 
Results:
None of the colony PCR's gave the correct size DNA piece.
 
[[IGEM:Harvard/2007/GeorgeXu/070719ColonyPCRgx|Gel]]
 
====Miniprep the RFP+JT====
I miniprepped the RFP+JT sample (originally for transformation in Top10 because BL21 expresses LacI, but we decided that we liked the idea of inducible RFP).
 
===7/18/07===
====Liquid Cultures====
I grew liquid cultures of the "correct" RFP+JT colony resulting from the colony PCR.
 
====Colony PCR of the tetR+JT and RFP+JT constructs====
I colony PCR'ed the teR+JT and RFP+JT constructs.
 
Results: Only one colony seemed correct. It was one of the constitutive RFP+JT parts.
 
[[IGEM:Harvard/2007/GeorgeXu/070718ColonyPCRgx|Gel]]
 
===7/17/07===
====Ligation and Transformation of tetR-JT and RFP-JT constructs====
I Roche ligated the following:
*([http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]) <([http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039] <[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002])
*[http://parts.mit.edu/registry/index.php/Part:BBa_J04450 J04450] <([http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039] <[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002])
 
and then transformed them into BL21.
 
[[IGEM:Harvard/2007/Protocols#Ligation|Ligation Protocol]]
[[IGEM:Harvard/2007/Protocols#Trasnformation|Transformation Protocol]]
 
====Clonewell and Vacufuge of the R-P constructs====
I clonewelled and vacufuged the R-P constructs and the JT. Unfortunately, I forgot to include the JT with the initial clonewell, so I ended up having to fish the very nasty clonewell out of the garbage (there was a piece of gel on it and chunks of gel were stuck in some of the wells). I used the last two wells (which were thankfully somewhat clean) for ladder and the JT. Unfortunately I ran into a few problems. First, the JT is about 2800 bp and the plasmid is about 2000 bp. The two bands on the clonewell were pretty close and it was difficult to separate the two bands sometimes. Second, the gel stopped running after a while. I noticed bubbles forming along the edges of the gel and whenver I pressed "Go," the gel would make an unhappy beeping noise and blink yellow, so I just stopped running the clonewell
 
[[User:GeorgeXu#My_First_Clonewell|Protocol]]
 
====Dephosphorylating the R-P constructs====
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Construct'''
| align="center" style="background:#f0f0f0;"|'''DNA'''
| align="center" style="background:#f0f0f0;"|'''AP Buffer'''
| align="center" style="background:#f0f0f0;"|'''AP'''
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #2||40 µL||4.7 µL||2 µL
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140] colony #1||42 µL||4.9 µL||2 µL
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1||36 µL||4.2 µL||2 µL
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140] colony #2 Lower Right||40 µL||4.7 µL||2 µL
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1 Upper Right||40 µL||4.7 µL||2 µL
|}
 
I then incubated at 37dC for 1 hour and heat inactivated at 65dC for 10 minutes.
 
[[IGEM:Harvard/2007/Protocols/Vector_Dephosphorylation_Protocol|Protocol]]
 
====Sequencing the R-P constructs====
I prepared and sent off reaction mixes. A summary of the order below:
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Label'''
| align="center" style="background:#f0f0f0;"|'''Construct'''
| align="center" style="background:#f0f0f0;"|'''Primer'''
|-
| AV01||J-hisGFPmek_5_1||VF2
|-
| AV02||J-hisGFPmek_5_1||VR
|-
| AV03||J_hisGFPmek_8_1||VF2
|-
| AV04||J_hisGFPmek_8_1||VR
|-
| AV05||[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #2||VF2
|-
| AV06||[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #2||VR
|-
| AV07||[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140] colony #1||VF2
|-
| AV08||[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140] colony #1||VR
|-
| AV09||[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1||VF2
|-
| AV10||[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1||VR
|-
| AV11||[http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140] colony #2 Lower Right||VF2
|-
| AV12||[http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140] colony #2 Lower Right||VR
|-
| AV13||[http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1 Upper Right||VF2
|-
| AV14||[http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1 Upper Right||VR
|}
 
====Digesting JT and R-P constructs====
I digested the following for 99:99 minutes:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Construct'''
| align="center" style="background:#f0f0f0;"|'''DNA'''
| align="center" style="background:#f0f0f0;"|'''Enzyme 1'''
| align="center" style="background:#f0f0f0;"|'''Enzyme 2'''
| align="center" style="background:#f0f0f0;"|'''Buffer 2'''
| align="center" style="background:#f0f0f0;"|'''BSA'''
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #2||40 µL||1 µL XbaI||1 µL PstI||4.7 µL||.5 µL
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140] colony #1||33 µL||1 µL SpeI||1 µL PstI||4.0 µL||.5 µL
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1||35 µL||1 µL SpeI||1 µL PstI||4.2 µL||.5 µL
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140] colony #2 Lower Right||30 µL||1 µL SpeI||1 µL PstI||3.6 µL||.5 µL
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1 Upper Right||33 µL||1 µL SpeI||1 µL PstI||4.0 µL||.5 µL
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039] <[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002]||33 µL||1 µL SpeI||1 µL PstI||4.0 µL||.5 µL
|}
 
====Miniprep and Nanodrop of JT and the R-P constructs====
I miniprepped the following parts:
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #2
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140] colony #1
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140] colony #2 Lower Right
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1 Upper Right
*[http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039] <[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002]
* 5
* 8
 
The "5" and "8" were Perry's samples. Note that the [http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1 Lower Right is missing. That is because the overnight culture didn't seem to grow. After centrifuging several times, there was still no pellet, so I just tossed that sample out.
 
The Nanodrop results (after Nanodropping, I made the following dilutions for sequencing samples):
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Construct'''
| align="center" style="background:#f0f0f0;"|'''Concentration'''
| align="center" style="background:#f0f0f0;"|'''Dilution'''
| align="center" style="background:#f0f0f0;"|'''µL of DNA'''
| align="center" style="background:#f0f0f0;"|'''µL of Water'''
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #2||97.6 ng/µL||1 in 2||10||10
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140] colony #1||173.2 ng/µL||1 in 4||5||15
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1||110.3 ng/µL||1 in 2||10||10
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140] colony #2 Lower Right||133.7 ng/µL||1 in 3||7||14
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1 Upper Right||132.8 ng/µL||1 in 3||7||14
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039] <[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002]||98.8 ng/µL||none||-||-
|-
| 5||112.1 ng/µL||1 in 2||10||10
|-
| 8||148.9 ng/µL||1 in 3||7||14
|}
 
[[IGEM:Harvard/2007/Protocols/Plasmid_MiniPrep_Protocol|Miniprep Protocol]]
[[IGEM:Harvard/2007/Protocols/NanoDrop%C2%AE_ND-1000_Protocol|Nanodrop Protocol]]
 
===7/16/07===
====Liquid Cultures====
I grew up liquid cultures of the following constructs, which come from the colony PCR's that Stephanie did in the morning:
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #2
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140] colony #1
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140] colony #2 Lower Right
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1 Upper Right
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1 Lower Right
*[http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039] <[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002]
 
The Upper/Lower Right is due to the fact that the colony PCR plate had duplicates of those colonies and it was unclear which colony was correct (at least, the PCR and gel was correct). So, I took both samples and labeled one as coming from the upper right of the plate (when viewed with the handwriting going the correct way) versus lower right.
 
====Dephosphorylation of J04450 and JT====
I used Antarctive Phosphatase to dephosphorylate the [http://parts.mit.edu/registry/index.php/Part:BBa_J04450 J04450] and the [http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039]>[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002]. I also heat inactivated at 65dC for 15 minutes.
 
After doing this, I was informed by Perry that I shouldn't dephosphorylate the insert. ARGH! I'll need to regrow, miniprep, and digest the JT.
 
[[IGEM:Harvard/2007/Protocols/Vector_Dephosphorylation_Protocol|Protocol]]
 
====Sequencing R0011, R0051, R0052====
I prepared the [http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011], [http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051], and [http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] for sequencing. They are AV09, AV10, and AV11 respectively. Stephanie and I dropped them off in the mailbox.
 
====Double Digest of J04450 and JT====
I used the following digest mixes:
*[http://parts.mit.edu/registry/index.php/Part:BBa_J04450 J04450]
**25 µL DNA
**1 µL SpeI
**1 µL PstI
**0.5 µL BSA
**3 µL Buffer 2
*[http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039]>[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002]
**20 µL DNA
**1 µL XbaI
**1 µL PstI
**0.5 µL BSA
**2.3 µL Buffer 2
 
[[IGEM:Harvard/2007/Protocols#Double_digest|Protocol]]
 
====Miniprep and Nanodrop of J04450 and JT====
I miniprepped and nanodropped the [http://parts.mit.edu/registry/index.php/Part:BBa_J04450 J04450] and the [http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039]>[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002].
 
Nanodrop results:
* [http://parts.mit.edu/registry/index.php/Part:BBa_J04450 J04450] - 91.1 ng/µL
* [http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039]>[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002] - 110.8 ng/µL
 
[[IGEM:Harvard/2007/Protocols/Plasmid_MiniPrep_Protocol|Miniprep Protocol]]
[[IGEM:Harvard/2007/Protocols/NanoDrop%C2%AE_ND-1000_Protocol|Nanodrop Protocol]]
 
===7/15/07===
====Liquid cultures====
I grew up liquid cultures of J04450 and JT in preparation for miniprep, digest, ligation, etc. on Monday 7/16/07.
====Transformation of constitutive tetR constructs: Take Two====
I transformed the
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011]+[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0051]+[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0052]+[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011]+[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0340]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0051]+[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0340]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0052]+[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0340]
into Top10 and plated on LB carb plates.
 
This was done in preparation for making the constitutive tetR+JT construct.
 
[[IGEM:Harvard/2007/Protocols#Transformation|Protocol]]
 
====Ligation of P0140, P0340, R0011, and R0051: Take Two====
I Roche ligated the following parts:
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011]+[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0051]+[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0052]+[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011]+[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0340]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0051]+[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0340]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0052]+[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0340]
 
This was done in preparation for making the constitutive tetR+JT construct.
 
[[IGEM:Harvard/2007/Protocols#Ligation|Protocol]]
 
====Vacufuge of R0011 and R0051: Take Two====
There were about .3 mL of water left in the the [http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011], [http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051], and [http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052], so I vacufuged for about 2 hours and then resuspended in 20 µL nuclease-free water.
 
This was done in preparation for making the constitutive tetR+JT construct.
 
====Clonewell of R0011 and R0051: Take Two====
I clonewelled the [http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011], [http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051], and [http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052].
 
This was done in preparation for making the constitutive tetR+JT construct.
 
[[User:GeorgeXu#My_First_Clonewell|Protocol]]
 
====Dephosphorylation of R0011 and R0051: Take Two====
After discovering that the colony PCR's of the BL21 transformations were horrible, Perry decided we should try it again, but dephosphorylate before we clonewell. So I dephosphorylated the [http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011], [http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051], and [http://parts.mit.edu/registry/index.php/Part:BBa_R0052 R0052] with the following mix:
* 38 µL DNA
* 3 µL Antarctic Phosphatase
* 4.6 µL Antarctic Phosphatase Buffer
 
This was done in preparation for making the constitutive tetR+JT construct.
 
[[IGEM:Harvard/2007/week_1#Vector_Dephosphorylation_Protocol_Again|Protocol]]
 
===7/13/07===
====Transformation of constitutive tetR constructs====
I transformed the following ligated parts into BL21:
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340]
 
This was done in preparation for making the constitutive tetR+JT construct.
 
[[IGEM:Harvard/2007/Protocols#Transformation|Protocol]]
 
====Ligation of P0140, P0340, R0011, and R0051====
I ligated the combinations of parts:
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340]
with the Roche Ligation Kit (the small white box with the tubes numbered 1-3)
 
Protocol:
#3 µL vector (R0051 and R0011) + 7 µL insert (P0140 and P0340)
#Vortex
#10 µL Vial 1 (ligation buffer)
#Vortex
#1 µL Vial 3 (ligase)
#Vortex, Spin down
#Let stand for 5 min at room temperature
 
This was done in preparation for making the constitutive tetR+JT construct.
 
[[IGEM:Harvard/2007/Protocols#Ligation|Protocol]]
 
====Dephosphorylation of R0011 and R0051====
I treated the R0011 and R0051 with Antarctic Phosphatase. My mix was:
*R0011
** 23.2 µL gel extracted R0011
** 2.7 µL Antarctic Phosphatase Buffer
** 1 µL Antarctic Phosphatase
*R0051
** 22.3 µL gel extract R0051
** 2.6 µL APB
** 1 µL AP
I then incubated for 1 hr @ 37dC and heat inactivated for 10 min @ 65dC.
 
This was done in preparation for making the constitutive tetR+JT construct.
 
[[IGEM:Harvard/2007/week_1#Vector_Dephosphorylation_Protocol_Again|Protocol]]
 
====Vacufuge of P0140, P0340, R0011, and R0051====
I vacufuged the samples digested on 7/12/07 for a total of about 3 hours. I then resuspended the samples in about 20 µL of nuclease-free water. I nanodropped them, but the numbers were ridiculously high (~200 ng/µL) and the curves looked awful. Note to self: never Nanodrop gel extraction again.
 
This was done in preparation for making the constitutive tetR+JT construct.
 
===7/12/07===
====My First Clonewell====
#Get iBase+Clonewell gel. Remove gel (leave the comb!) and place into iBase.
#Pre-run (Mode 0) for two minutes while the comb is still in the gel
#Take out comb, load each well in top row with 20 µL samples, the middle well with ~10 µL ladder, and the bottom row wells with 20 µL nuclease-free water
#Run the gel with (Mode 5: Run Clonewell), watch the bands and stop the gel when your desired band hits the dashed line above the bottom row of wells
#With the gel stopped, fill the bottom row wells with nuclease-free water.
#Run the gel again. When you see the band starting to enter the well, take out the sample in the well and put into clean labeled centrifuge tube. Then refill the well with 20 µL nuclease-free water and continue running the gel.
#Repeat this procedure until you get enough sample. If the band runs past the well, you can use Mode 6: Reverse Clonewell to make the band move back into the well
 
So basically, I did this for the stuff that was digested (see immediately below) and I ended up with a lot of water, so I am going to vacufuge the results tomorrow.
 
This was done in preparation for making the constitutive tetR+JT construct.
 
====Digests of P0140, P0340, R0011, and R0051====
*XbaI and PstI digests
**[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340]
**[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140]
*SpeI and PstI digest
**[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011]
**[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051]
*Mixture
**42.5 µL DNA
**1 µL enzyme 1
**1 µL enzyme 2
**0.5 µL BSA
**5 µL Buffer 2
 
This was done in preparation for making the constitutive tetR+JT construct.
 
[[IGEM:Harvard/2007/Protocols#Double_digest|Protocol]]
 
====Perry's E-gel====
I ran an E-gel for Perry's colony PCR.
#Take out E-gel (leave comb in) and put in one of the bases. NOTE: One of the red bases has a faulty contact which requires covering by aluminum foil to work correctly.
#Pre-run the gel for 2 minutes. (Press and hold either 15min or 30min on the red bases or Mode 0 on the gray bases)
#When the pre-run is finished, press any button to stop the beeping.
#Load 10 µL of nuclease-free water and 10 µL of ladder into the first well. Then 10 µL nuclease-free water + 10 µL sample into every other well. If there are empty wells, put in 20 µL of nuclease-free water into the empty wells. NOTE: Nick mentioned this and it is a pretty good idea. Put the ladder in the middle well or if there are empty wells, put ladder in the end empty wells. This way, it is easier to compare, especially if the bands bend.
#Run the E-gel for 30-min (Press 30min on the red bases, or run Mode 1 - changing the time if necessary)
#When the run is done, press any button to stop the beeping. Use the UV machine in the small room to visualize the gel.
 
It confirmed that P0140 and P0340 were correct. However, I don't think his GFPmeks turned out very well.
 
[[IGEM:Harvard/2007/George/Perry_Egel_07_11_07|Results]]
 
===7/11/07===
====Sequencing====
I sent out the following for Genewiz sequencing:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Nanodrop (ng/µL)'''
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_F2620 F2620] > [http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]||130
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_I13273 I13273]||150
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039] > [http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002]||140
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608] > [http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]||220
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_S03623 S03623] > [http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]||190
|-
| [http://parts.mit.edu/registry/index.php/Part:BBa_S03623 S03623] > [http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]||75
|}
 
The plan was to dilute the above samples to about 40-60 ng/µL and I did the dilutions, but I stupidly ended up using the stock instead of the dilution so our samples had a LOT more DNA than we had planned. Eh, it still ended up working out.
 
[[IGEM:Harvard/2007/Protocols#Sequencing|Protocol]]
 
Order Summary:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Tube Label'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Primer'''
|-
| AV01||[http://parts.mit.edu/registry/index.php/Part:BBa_S03623 S03623] > [http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]||VF2
|-
| AV02||[http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608] > [http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]||VF2
|-
| AV03||[http://parts.mit.edu/registry/index.php/Part:BBa_S03623 S03623] > [http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]||VF2
|-
| AV04||[http://parts.mit.edu/registry/index.php/Part:BBa_F2620 F2620] > [http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]||VF2
|-
| AV05||[http://parts.mit.edu/registry/index.php/Part:BBa_I13273 I13273]||VF2
|-
| AV06||[http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039] > [http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002]||VF2
|-
| AV07||[http://parts.mit.edu/registry/index.php/Part:BBa_S03623 S03623] > [http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]||VR
|-
| AV08||[http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608] > [http://parts.mit.edu/registry/index.php/Part:BBa_I13507 I13507]||VR
|-
| AV09||[http://parts.mit.edu/registry/index.php/Part:BBa_S03623 S03623] > [http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]||VR
|-
| AV10||[http://parts.mit.edu/registry/index.php/Part:BBa_F2620 F2620] > [http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]||VR
|-
| AV11||[http://parts.mit.edu/registry/index.php/Part:BBa_I13273 I13273]||VR
|-
| AV12||[http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039] > [http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002]||VR
|}
-------
 
===7/10/07===
[[Shlo/notebook#Colony_PCRs|Colony PCRs]]
 
===7/9/07===
====Growing Liquid Cultures of Tomorrow's Plate Reader Samples====
[[IGEM:Harvard/2007/Laboratory_Notebooks/Quorum_Sensing/Week_2#Growth_of_colonies_in_liquid_cultures|Protocol]]
 
Stephanie and I grew liquid cultures of the following parts:
*[http://parts.mit.edu/registry/index.php/Part:BBa_I13263 I13263]
*[http://parts.mit.edu/registry/index.php/Part:BBa_I13273 I13273]
*[http://parts.mit.edu/registry/index.php/Part:BBa_I5311 I5311]
*[http://parts.mit.edu/registry/index.php/Part:BBa_I13522 I13522]
*[http://parts.mit.edu/registry/index.php/Part:BBa_B0015 B0015]
*[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002 (+)]
*[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002 (-)]
Also, Stephanie left the T9002 (+) sample incubating with 1000 nM OHHL to serve as a comparison to J37015.
 
====Colony PCR of Ligated Parts====
[[IGEM:Harvard/2007/Protocols#Colony_PCR.2Fplate_streak|Protocol]]
 
Stephanie, Perry, and I colony PCR'ed the parts that they ligated the week before.
 
See [[Shlo/notebook#7.2F9|Stephanie's notebook]] for results.
===6/29/07 FACS DAY (I13263)!===
====FACS-related====
*8:30 AM - Stephanie and I took the OD of the overnight cultures.
**Results - 1.785, 1.793, 1.780
*9:15 AM - Stephanie and I performed first dilution (growing the 4 hr induction cells). Unfortunately, we didn't dilute correctly...
**1:40 dilution: 50 µL liquid culture + 148 µL LB broth + 2 µL ampicillin
**1:80 dilution: 25 µL liquid culture + 173 µL LB broth + 2 µL ampicillin
**1:100 dilution: 20 µL liquid culture + 178 µL LB broth + 2 µL ampicillin
*10:30 AM - Oopsie, we realized we didn't dilute 1:40, etc.; instead, we had actually diluted 1:4, etc. We measured the OD's of these incorrectly diluted samples and rediluted the samples. Note that in order to measure the OD, we diluted samples 1:100, measured the OD, and multiplied the measured OD by 100 to give us our measurement for the actual sample.
**1:4 sample OD = 3, redilute 1:100 giving us a sample of about OD=0.03
**1:8 sample OD = 1.8, redilute 1:30 giving us a sample of about OD=0.05
**1:10 sample OD = 1.3, redilute 1:100 giving us a sample of about OD=0.01
**Also, we diluted 1:40, 1:80, and 1:100 2 mL samples from the overnight stock to grow as our 2 hr induction cells
*11:00 AM - I diluted the stock solution 1:10,000 and 1:1,000,000 in 100 µL 200-proof ethanol to give a 97 µM and 97 nM solution.
*11:30 AM - Stephanie and I diluted 1:40, 1:80, and 1:100 2 mL samples from the overnight stock to grow as our 1 hr induction cells
*12 noon - Stephanie and I took the OD of the 4 hr induction cells
**4 hr induction cells, 1:40: 0.2
**4 hr induction cells, 1:80: 0.2
**4 hr induction cells, 1:80: 0.1
Since each of these were about 2 mL samples, we didn't have enough sample to have 2 mL cuvette samples. Instead, we used 1mL of cells with 10.5 uL HSL. 2 samples were taken from the 1:40 first dilution (8:30am) - used for the 10 and 100nM HSL runs - and 1 from the 1:80, used with 1nM HSL.
*1:30 pm - Took OD of the 1hr and 2hr samples + Induce the 2 hr samples
 
See [[Shlo/notebook#6.2F29|Stephanie's entry]] for the rest
====Transformation and Liquid Culture of Assorted BioBricks====
[[IGEM:Harvard/2007/Protocols#Transformation|Transformation Protocol]]
 
[[IGEM:Harvard/2007/Laboratory_Notebooks/Quorum_Sensing/Week_2#Growth_of_colonies_in_liquid_cultures|Liquid Cultures Protocol]]
 
Perry and I transformed and prepared liquid cultures of the following parts:
*[http://parts.mit.edu/registry/index.php/Part:BBa_S03623 S03623]
*[http://parts.mit.edu/registry/index.php/Part:BBa_S03608 S03608]
*[http://parts.mit.edu/registry/index.php/Part:BBa_J37034 J37034]
*[http://parts.mit.edu/registry/index.php/Part:BBa_J23039 J23039]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0140 R0140]
*[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002]
*[http://parts.mit.edu/registry/index.php/Part:BBa_B0015 B0015]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051]
 
===6/28/07===
====Resuspending the HSL stock====
I took the 25 mg of [http://www.sigmaaldrich.com/catalog/search/ProductDetail/FLUKA/09945 N-Butyryl-DL-homoserine lactone] and resuspended in 1.5 mL of 200-proof ethanol. I then put the tube in the Quorum sensing box in the freezer at Stephanie's bench.
 
This was done in preparation for inducing the FACS samples on 6/29/07.
====Digestion of F1610: Part Two====
Stephanie ran another F1610 digest with
#2.5 µL Buffer 2
#0.5 µL BSA
#0.5 µL XbaI
#0.5 µL PstI
#21 µL F1610
 
Basically it is the same as [[User:GeorgeXu#Digestion_of_F1610_.28XbaI.2C_PstI.29|the previous digestion]] except we now have 21 µL vs. 15 or 6 µL of DNA. This was done because the Clonewell of the F1610 that Perry and I ran had an almost invisibly faint band corresponding to the F1610. Where did all the DNA go?
====Grow Liquid Cultures of I13263====
[[IGEM:Harvard/2007/Laboratory_Notebooks/Quorum_Sensing/Week_2#Growth_of_colonies_in_liquid_cultures|Protocol]]
 
Stephanie and I grew I13263 in preparation for the FACS on 6/29/07.
====Sequences====
Perry checked the sequences that we got back from Genewiz<br>
Results<br>
* [http://parts.mit.edu/registry/index.php/Part:BBa_F1610 F1610] (something is very wrong...)
** The actual part is 798 bp, we got back a sequence ~300 bp
** The sequence contains the terminator and some random junk after it
* [http://parts.mit.edu/registry/index.php/Part:BBa_I13263 I13263] (w00t)
** The first and last ~650 bp are perfect.
* [http://parts.mit.edu/registry/index.php/Part:BBa_I13272 I13273]
** !!!!add something here
!!!!upload the sequences?
 
====Clonewell of F1610====
[User:GeorgeXu#My_First_Clonewell Protocol]
 
Results
 
[http://parts.mit.edu/registry/index.php/Part:BBa_F1610 F1610] is about 800 bp. There is an almost invisibly faint band at 800 bp. What happened to all the DNA? We need to run it again
 
====Transformation of Additional Parts====
[[IGEM:Harvard/2007/Protocols#Transformation|Protocol]]
 
Perry and I transformed the following parts
 
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0040 R0040]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051]
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011]
*[http://parts.mit.edu/registry/index.php/Part:BBa_E0240 E0240]
*[http://parts.mit.edu/registry/index.php/Part:BBa_B0015 B0015]
*[http://parts.mit.edu/registry/index.php/Part:BBa_T9002 T9002]
 
====Digestion of F1610 (XbaI, PstI)====
 
Mix 1:
#2.5 µL Buffer 2
#0.5 µL BSA
#0.5 µL XbaI
#0.5 µL PstI
#15 µL F1610 (168 ng/µL)
#6 µL Nuclease-free water
Mix 2:
#2.5 µL Buffer 2
#0.5 µL BSA
#0.5 µL XbaI
#0.5 µL PstI
#15 µL Nuclease-free water
#6 µL F1610 (168 ng/µL)
 
Perry and I ran two digests of F1610 with different amounts of DNA. The plan is to gel extract and ligate with the promoters that Perry has transformed.
 
[[IGEM:Harvard/2007/Protocols#Double_digest|Protocol]]
 
===6/27/07===
====Sequencing the Midiprepped QS parts====
[[IGEM:Harvard/2007/week_2#Sequencing_DNA|Protocol]]
 
Just checking if the Midiprepped sequences are correct. Details of the order:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Tube Label'''
| align="center" style="background:#f0f0f0;"|'''DNA Name'''
| align="center" style="background:#f0f0f0;"|'''DNA Length'''
| align="center" style="background:#f0f0f0;"|'''Primer'''
|-
| AV01||F1610||3987||VF2
|-
| AV02||F1610||3987||VR
|-
| AV03||I13263||4123||VF2
|-
| AV04||I13263||4123||VR
|-
| AV05||I13272||4162||VF2
|-
| AV06||I13272||4162||VR
|}
====Transformation of I13263====
[[IGEM:Harvard/2007/week_1#Transformation_protocol|Protocol]]
 
Stephanie and I transformed 1 µL of the I3263 that we midiprepped into 30 µL of BL21. We then plated them onto agar+ampicillin plates. We eventually want to carry out a test experiment by inducing the I3263 with different concentrations of HSL to test and possibly start characterizing the part.
 
====Hispeed! Midiprepping of the QS parts====
[[IGEM:Harvard/2007/Protocols/HiSpeed_Midi_and_Maxi_Prep|Protocol]]
 
Perry and I Midiprepped the QS parts in order to get DNA that we could sequence and transform.
 
===6/26/07===
 
====Growth of QS parts in Liquid Cultures====
[[IGEM:Harvard/2007/Laboratory_Notebooks/Quorum_Sensing/Week_2#Growth_of_colonies_in_liquid_cultures|Protocol]]
 
We grew the QS parts in a liquid LB+ampicillin broth.
====Nanodrop====
[[IGEM:Harvard/2007/Protocols/NanoDrop%C2%AE_ND-1000_Protocol|Protocol]]
 
Results<br>
10mer: 389.8 ng/µL
15mer: 278.6 ng/µL
20mer: 168.7 ng/µL
====Nucleotide Removal of the 10mer, 15mer, and 20mer library====
Again, this was done in preparation for the ligation reaction that we will do with the massive amounts of DNA.
 
[[IGEM:Harvard/2007/week_2#Nucleotide_Removal_3|Protocol]]
 
====PCR Extension of the 10mer, 15mer, and 20mer library====
Basically, this was done in preparation for the ligation reaction that we will do with the massive amounts of DNA.
 
[[IGEM:Harvard/2007/week_2#Digestion_of_the_10mer.2C_15mer.2C_and_20mer_Library.2C_along_with_vectors_from_week_0|Protocol]]
 
===6/25/07===
====Transforming and Plating the QS parts====
[[IGEM:Harvard/2007/Protocols#Transformation|Protocol]]
 
Perry transformed the parts [[F1610]], [[I13263]], and [[I13272]] into Top10 cells and plated them on LB+ampicillin plates. We want to sequence the parts, test them, and eventually use them in our BL21 cells.
 
===6/22/07===
==== Counting colonies: Overnight vs. Short Ligation ====
We counted the number of colonies in the Overnight vs. Short Ligation plates.<br>
[[IGEM:Harvard/2007/Plates|Results]]
 
==== Nanodrop of Ligated Colonies====
[[IGEM:Harvard/2007/Protocols/NanoDrop%C2%AE_ND-1000_Protocol|Protocol]]
 
Results<br>
OmpA1+random library+extension: 50.8 ng/µL
OmpA2+random library+extension: 50.7 ng/µL
The curves looked smooth. %
===6/21/07===
==== Protein Gel of OmpA+His/OmpA+Strep Cells ====
[[IGEM:Harvard/2007/week_1#Running_Protein_Gel_of_Induced_Bacteria_Outer_Membrane_Proteins|Protocol]]
 
We ran this protein gel to see if the OmpA1+His, OmpA1+Strep, OmpA2+His, and OmpA2+Strep was actually expressed. Also, we wanted to see if there was any difference between the immediate induction, delayed induction, and no induction.
 
[[IGEM:Harvard/2007/Gels#6.2F22_overexpression_gel|Results]]
 
==== Bacterial Innoculation and Induction ====
[[IGEM:Harvard/2007/week_1#Bacterial_Innoculation_and_Induction|Inocculation and Induction Protocol]]
 
[[IGEM:Harvard/2007/week_1#Measuring_Optical_Density_to_measure_cell_density|Measuring Optical Density Protocol]]
 
We took the OmpA1+His, OmpA1+Strep, OmpA2+His, and OmpA2+strep that were grown in liquid culture overnight starting on 6/20/07 and split them into three sets. The first set was not induced, the second set was immediately induced with 10 µL IPTG¸
and the final set was induced once it hit log phase, which we determined by measuring the optical density.
 
[[IGEM:Harvard/2007/week_1/absorbance|Optical Density Results]]
 
===6/20/07===
====Gel Extraction====
 
[[IGEM:Harvard/2007/week_1#Gel_Extraction|Protocol]]
 
We extracted the DNA from the gel we ran on 6/19/07
 
[[Gels#6.2F19_gel_after_excision|Gel after Excision]]
 
===6/19/07===
 
====Running the OmpA1/OmpA2 gel====
[[IGEM:Harvard/2007/week_1#Setting_up_and_running_gels|Protocol]]
 
We poured a gel and ran the DNA from the dephosphorylated samples.
 
[[IGEM:Harvard/2007/Gels#6.2F19_gel_after_excision|Results]]
 
====Dephosphorylating the OmpA1/OmpA2====
[[IGEM:Harvard/2007/week_1#Vector_Dephosphorylation_Protocol_Again|Protocol]]
 
Six times this amount of mix was made as a Master Mix in order to dephosphorylate the OmpA1 and OmpA2. There were five groups total:
#Group 1: OmpA1, Nhe1, Pst1
#Group 2: OmpA1, Nhe1, Pst1
#Group 3: OmpA2, Nhe1, Pst1
#Group 4: OmpA2, Nhe1, Pst1
#Group 5: Perry...

Latest revision as of 18:07, 20 December 2010

About Me

I am currently a graduate student in the joint program between Harvard Biophysics and HST.

Research History

2005 Summer

I studied Berry Shrivel Disease under the mentorship of Professor Ken Shackel and Dr. Mark Krasnow at UC Davis's Department of Viticulture and Enology through the Young Scholar's Program. I imaged grape berry sections bathed in fluorescein diacetate, which is intracellularly hydrolyzed by viable cells to the fluorescent protein fluorescein [1], in order to assess cell viability in diseased grapes. Our hypothesis was that the disease was caused by massive cell death, in particular the companion cells surrounding the phloem. I had to leave for school before distinct symptoms presented and my data could be analyzed, but later analysis proved inconclusive. I can no longer find the CD with all of my 1000+ awesome images, but you can see a sample on slide 23 of Dr. Krasnow's presentation.

2007 Spring

During the second semester of my freshman year, I briefly worked part-time in the Weitz Lab with Dr. Amy Rowat on a microfluidics device for fluorescent detection, actuation, and control of individual yeast cells for directed evolution studies.

2007 Summer - 2007 Winter

During the summer following my freshman year I was a member of Harvard's 2007 iGEM team. The overall goal of our project was to have bacteria that would bind to a target and activate a signal, either through quorum sensing or transmembrane signal transduction. Our project consisted of three modules:

  1. We planned to utilize surface expression of a peptide library through fusion with the surface proteins Lpp-OmpA [2] and autodisplay protein AIDA-1 [3] for screening of binding sequences. We were able demonstrate successful fusion and targeting with his and strep2 tags but did not have time to test a random library.
  2. The project that I helped brainstorm was the quorum sensing. We utilized LuxI/LuxR quorum sensing found in V. fishcheri to create two systems: one was a single-cell that created both the "sender" LuxI molecule and the "receiver" luxR molecule, while the other had the "sender" and "receiver" constructs in separate cells. We successfully demonstrated quorum sensing in our system and began tests of combining these constructs with surface expression. My work on this project is (mostly) documented in my iGEM Lab Notebook.
  3. We planned to construct transmembrane signal transduction using the Fec system [4]. Unfortunately, the project turned out to be too ambitious and we were unsuccessful.

Professor George Church was one of the faculty advisors for this program and one of his students, Harris Wang, was one of the Teaching Fellows. After the iGEM jamboree, I began working for Harris in the Church lab.

2008 Summer

During the summer following my sophomore year I received generous funding from the Harvard Reischauer Institute's Summer Internship Program to work in Japan for 10 weeks. While there, I prepared and published a review article under the mentorship of Masakazu Umezawa and Professor Ken Takeda on the permanent health effects of adverse fetal environments.

2008 Spring - 2010 Spring

I worked in the Church lab with Harris Wang and Dr. Farren Isaacs on lambda-red mediated recombineering [5]. Most of my work is documented on an internal lab wiki.

Publications

  1. Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, Forest CR, and Church GM. 'Programming cells by multiplex genome engineering and accelerated evolution'. Nature. 460, 894 (2009)

References

  1. Heslop-Harrison J and Heslop-Harrison Y. Evaluation of pollen viability by enzymatically induced fluorescence; intracellular hydrolysis of fluorescein diacetate. Stain Technol. 1970 May;45(3):115-20. DOI:10.3109/10520297009085351 | PubMed ID:4192549 | HubMed [FDA]
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All Medline abstracts: PubMed | HubMed

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