IGEM:IMPERIAL/2007/Notebook/2007-8-22: Difference between revisions

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__NOTOC__
==Midiprep of Biobricks==
==Midiprep of Biobricks==
Midipreped 7 Biobricks  
Midipreped 6 Biobricks  


<font size=-2>
<font size=-2>
*BBa_I13522 [ptet-GFP]
*2. BBa_I13522 [ptet-GFP]
*BBa_R0051 [pcI]
*21. BBa_J23039 [ptet-luxI]
*BBa_J23039 [ptet-luxI]
*23. BBa_I13504 [GFP]
*BBa_I13504 [GFP]
*24. BBa_R0062 [pLux (no luxR)]
*BBa_R0062 [pLux (no luxR)]
*25. BBa_J37032 [pLux-GFP]  
*BBa_J37032 [pLux-GFP]  
*26. BBa_S03119 [tet-LuxR]</font>
*BBa_S03119 [tet-LuxR]</font>


Protocols can be found at [[IGEM:IMPERIAL/2007/Notebook/General_Protocols#Midiprep|Midiprep]] in the general protocols page
Protocols can be found at [[IGEM:IMPERIAL/2007/Notebook/General_Protocols#Midiprep|Midiprep]] in the general protocols page
Line 23: Line 22:
==Miniprep of Biobrick==
==Miniprep of Biobrick==
Minipreped 2x1 Biobrick <font size=-2>
Minipreped 2x1 Biobrick <font size=-2>
*BBa_R0051 [pcI]</font>
*14. BBa_R0051 [pcI]</font>


Protocols can be found at [[IGEM:IMPERIAL/2007/Notebook/General_Protocols#Miniprep|Miniprep]] in the general protocols page
Protocols can be found at [[IGEM:IMPERIAL/2007/Notebook/General_Protocols#Miniprep|Miniprep]] in the general protocols page
==Restriction Digest of Biobricks==
#4 DNA samples were digested by EcoRI and PstI
#* Samples 2, 14a, 14b, 24
#4 DNA samples were digested by Xbai
#* Samples 21, 23, 25, 26
#4 μl of DNA was digested by 1 μl of enzymes at 37&deg;C for 1 hour


==Agarose Gel Electrophoresis of Biobricks==
==Agarose Gel Electrophoresis of Biobricks==
#Checked 6 Biobricks on 1% agarose gel  
#Checked 7 Biobricks and 7 Digests on 1% agarose gel  


Protocols can be found at Protocols can be found at [[IGEM:IMPERIAL/2007/Notebook/General_Protocols#Electrophoresis|Electrophoresis]] in the general protocols page
Protocols can be found at Protocols can be found at [[IGEM:IMPERIAL/2007/Notebook/General_Protocols#Electrophoresis|Electrophoresis]] in the general protocols page


<br> Pictures of the gel to be put up soon
[[Image:ICGEMS Gel 22 8.jpg]]
 
Conclusions:
*Part 14 is confirmed to be faulty
*Part 21 does not have the right size


==In Vivo Testing of pT7 (Continued)==
==In Vivo Testing of pT7 (Continued)==
#Continue testing for fluorescence reading of pT7 in the cell sample left in the 37<sup>o</sup>C incubator overnight.
Continue testing pT7 in vivo at 37<sup>o</sup>C. This test was started on 21-08-2007 and has been left over night.
<br><br>
Protocol can be found here under [[IGEM:IMPERIAL/2007/Experimental_Design/Phase1/Protocol_1.1|Phase 1-In vivo testing]] on the experimental design page.
<br><br>
Results can here under [[IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results 2.1 | Results]] on the experimental design page


==In Vitro Testing of pTet 37<sup>o</sup>C (Continued)==
==In Vitro Testing of pTet 37<sup>o</sup>C (Continued)==
#Continued Testing for fluorescence reading from the in vitro plate left over from yesterday.
Continued with the testing of pTet in vitro which was started on 21-08-2007 at 11.30pm. This experiment will carry on until fluorescence reaches that of the control.
<br><br>
Protocol can be found here under [[IGEM:IMPERIAL/2007/Experimental_Design/Phase1/Protocol_2.1|Phase 1-In vitro testing]] on the experimental design page.
<br><br>
Results can here under [[IGEM:IMPERIAL/2007/Experimental_Design/Phase1/Results_2.1| Results]] on the experimental design page


==Protocol - In vitro testing - Operating Temperature Range and Initial Testing pT7 phase==
==In vitro Testing of pT7 37<sup>o</sup>C==
===Status===
Initial testing of pT7 in vitro at 37<sup>o</sup>C. This experiment will carry on until fluorescence reaches that of the control.
*All tests for opertating range were completeted and the intial pT7 test carried out
<br><br>
===Aims===
Protocol can be found here under [[IGEM:IMPERIAL/2007/Experimental_Design/Phase1/Protocol_2.1|Phase 1-In vitro testing]] on the experimental design page.
*To determine the exponential phase of production of GFP at extreme points of temperatures we are considering.
<br><br>
**10<sup>o</sup>C and 50<suP>o</sup>C for pTet in vitro
Results can here under [[IGEM:IMPERIAL/2007/Experimental_Design/Phase1/Results_2.1| Results]] on the experimental design page
**37<sup>o</sup>C for pT7 in vitro


===Equipments===
==Operating Range of in vitro Testing of pTet==
*Fluorometer + PC
Initial testing of operating ranges of pTet in vitro at 10<sup>o</sup>C and 45<sup>o</sup>C. The sampling of this was every 30minutes.
*Fridge at 4<sup>o</sup>C
<br><br>
*Water bath in cold room at 10<sup>o</sup>C
Protocol can be found here under [[IGEM:IMPERIAL/2007/Experimental_Design/Phase2/Protocol_2.1.1|Phase 2-Operating Temperature Ranges]] on the experimental design page.
*37<sup>o</sup>C shaking incubator
<br><br>
*50<sup>o</sup>C water bath
Results can here under ... on the experimental design page
*3 Fluorometer plates (black)
*6 Sealing plate mats
*Gilson pipettes 200, 20, 10
*Eppendorf Tubes
*Plate Centrifuge
*Stopwatch


===Reagents===
*Commercial S30 E.coli extract. Including:
**175µl Amino Acid Mixture Minus Cysteine, 1mM
**175µl Amino Acid Mixture Minus Methionine, 1mM
**175µl Amino Acid Mixture Minus Leucine, 1mM
**450µl S30 Extract, Circular (3 × 150µl)
**750µl S30 Premix Without Amino Acids
*Commercial S30 T7 extract. Including:
**175µl Amino Acid Mixture Minus Cysteine, 1mM
**175µl Amino Acid Mixture Minus Methionine, 1mM
**175µl Amino Acid Mixture Minus Leucine, 1mM
**450µl T7 S30 Extract, Circular (3 × 150µl)
**750µl S30 Premix Without Amino Acids
*MiiA water x1ml
*GFP solution (For this initial experiment does not need to be purified GFP, we just want to know we have the right filter and that our settings are adjusted to measuring GFP)


===Steps===
==Vesicles Formation with GFP==
#First collect all equipment and reagents and ensure that the fluorometer and that the PC connected has a data collection protocol installed.
*Place one of the 96 well plate in the 10oC water bath, another n the 37<sup>o</sup>C incubator, and the last one in the 45<sup>o</sup>C incubator. This is done so as to heat them up to the appropriate temperature before the experiments start.
#''Commercial E.coli Cell Extract'': Take out 3 tubes of each component of the cell extract prepared earlier. 
#''Commercial E.coli Cell Extract'': Pipette 20µl of each type of amino acid mix- AA without leucine + AA without cysteine, into an eppendorf tube. This is called the complete amino acid mixture.
#''Commercial E.coli Cell Extract'':Take another eppendorf tube and add 5µl of the E.coli complete amino acid mixture. Then add 20µl of S30 Premix Without Amino Acid. Then add 15µl of S30 Extract Circular. Place the eppendorf tube in a rack on the bench. Repeat the step for a total of 8 tubes.
#''Commercial E.coli T7 Cell Extract'': Repeat the above steps for the T7 E.coli cell extract. This time, take 2 tubes of prepared mixture; as well as prepare 10µl of amino acid mix each, to form 20µl of complete amino acid mixture.
#Vortex the tubes to mix thoroughly and place the 4 tubes of E.coli commercial extract in each incubator for ten minutes.
#Place 60μl of midipreped DNA plasmid (pT7) into an eppendorf tube and place it in the 37<sup>o</sup>C incubator.
#Place another 60μl of midipreped DNA plasmid (pTet) into two eppendorf tubes each and place one of them in the 10<sup>o</sup>C water bath, and the other in the 45<sup>o</sup>C water bath.
#Place 3 96 well plates into their respective incubators.


===Loading Plate===
{|align="right" border="1"
#Follow the schematic for the plate and begin by loading the in vitro expression system into the correct wells. Before loading in the samples vortex the tubes for a few seconds to mix the solution.
| width="200px"| [[image:IC07_image095.jpg|200px]]
#Place the lid on the 96well plate and tape up the edges of the lid. This should be put into the incubator at 37oC for 10 minutes to allow temperature to equilibrate.
| width="200px"| [[image:IC07_image096.jpg|200px]]
#Remove from incubators and spin-down in centrifuge in plate centrifuge at 2000rpm for a few seconds. Spin down is the process of bringing down any solution on lid or side of well into the base of the well. Alternatively can tap the top of the lid to bring down any solution to bottom of the well.
|-
#Remove lid off the 96 well plate and place in the fluorometer. Create a file name protocol 2-1 under: D:\IGEM\'''INSERT DATE'''\CBD\ OTR. Export the data here. If repeated measurements change the second number to suit repeat number, e.g. 2nd repeat protocol 2-2, 5th repeat protocol 2-5. Once the data collection is set up then initiate the measurements.
|colspan="2" width="400px"|Microscope pictures from sample 4 (2ml emulsion, centrifuge for 20min at 30x g). Left: A fluorescent object. Right: The same object under white light.
#This measurement will give a back ground fluorescence measurement and can be used as our time zero data.
|-
#Then to begin the reaction add 20μl of purified DNA sample to each well indicated on the schematic. Be careful not to add to wells that DO NOT NEED DNA.
| width="200px"| [[image:IC07_image086.jpg|200px]]
#Place lid back on and place back in the respective incubators.
| width="200px"| [[image:IC07_image089.jpg|200px]]
#After 30 minutes of incubation measure the fluorescence by repeating procedure 3-4 above. This initial measurement of 30 minutes is to find out how fast GFP is being produced. After this initial measurement, the intervals should be reassessed and adjusted accordingly.
|-  
#Before each measurement be careful to remember to either spin down or tap down the solution and to remove the lid before placing in the fluorometer.
|colspan="2" width="400px"|Strange structures. Left: Aggregates in the oil-lipid emulsion. Right: Air-water interface and an unexpected 'bubbly' structure in the aqueous region.
#Repeat the above steps until 6 hours after the DNA was first added.
|}
'''Formation of Vesicles'''


==Pilot Preparation of Vesicles==
Using a prepared well-dessicated, well sonicated DOPC/mineral oil suspension from [[IGEM:IMPERIAL/2007/Notebook/2007-8-21#Pilot Preparation of Vesicles | yesterday]],
'''Formation of Vesicles'''
* 2ml of suspension was taken to prepare an interface according to protocol.
* 10x diluted GFP solution was used to prepare the emulsion; stirred gently with magnetic stirrer.
* Special care was taken to protect the GFP solution from light at all times.
 
4 samples prepared:
* Sample 1: 2ml of emulsion, no centrifuge.
* Sample 2: Following the protocol, with 2ml suspension interface and 100&mu;l of emulsion added.
* Sample 3: 2ml of emulsion, centrifuge at 120x g for 10mins.
* Sample 4: 2ml of emulsion, centrifuge at 30x g for 20mins.




'''Results'''
'''Results'''
Light Microscopy (Magnification - 100x):
* Emulsion: Vesicles seen, presumably monolayer.
* Sample 1: Water-oil interface observed. Presumably the phospholipids have clumped altogether.
* Sample 2: Vesicles seen.
* Sample 3: Vesicles seen.
* Sample 4: Vesicles seen.
Fluorescence Microscopy (Magnification - 100x):
* Emulsion: GFP vesicles seen. Lots of GFP artifacts.
* Sample 1: No fluorescent vesicles observed.
* Sample 2: No fluorescent vesicles observed.
* Sample 3: "Vesicles" of sizes 1-10 &mu;m found encapsulating GFP within. Lots of GFP artifacts.
* Sample 4: "Vesicles" of sizes 1-10 &mu;m found encapsulating GFP within. Lots of GFP artifacts.




'''Preparations'''
'''Preparations'''
No preparations were carried out.

Latest revision as of 15:05, 23 October 2007

<calendar> name=iGEM:IMPERIAL/2007/Notebook date=2007/09/22 view=threemonths format=%name/%year-%month-%day weekstart=7 </calendar>

Midiprep of Biobricks

Midipreped 6 Biobricks

  • 2. BBa_I13522 [ptet-GFP]
  • 21. BBa_J23039 [ptet-luxI]
  • 23. BBa_I13504 [GFP]
  • 24. BBa_R0062 [pLux (no luxR)]
  • 25. BBa_J37032 [pLux-GFP]
  • 26. BBa_S03119 [tet-LuxR]

Protocols can be found at Midiprep in the general protocols page

Miniprep of Biobrick

Minipreped 2x1 Biobrick

  • 14. BBa_R0051 [pcI]

Protocols can be found at Miniprep in the general protocols page

Restriction Digest of Biobricks

  1. 4 DNA samples were digested by EcoRI and PstI
    • Samples 2, 14a, 14b, 24
  2. 4 DNA samples were digested by Xbai
    • Samples 21, 23, 25, 26
  3. 4 μl of DNA was digested by 1 μl of enzymes at 37°C for 1 hour

Agarose Gel Electrophoresis of Biobricks

  1. Checked 7 Biobricks and 7 Digests on 1% agarose gel

Protocols can be found at Protocols can be found at Electrophoresis in the general protocols page

Conclusions:

  • Part 14 is confirmed to be faulty
  • Part 21 does not have the right size

In Vivo Testing of pT7 (Continued)

Continue testing pT7 in vivo at 37oC. This test was started on 21-08-2007 and has been left over night.

Protocol can be found here under Phase 1-In vivo testing on the experimental design page.

Results can here under Results on the experimental design page

In Vitro Testing of pTet 37oC (Continued)

Continued with the testing of pTet in vitro which was started on 21-08-2007 at 11.30pm. This experiment will carry on until fluorescence reaches that of the control.

Protocol can be found here under Phase 1-In vitro testing on the experimental design page.

Results can here under Results on the experimental design page

In vitro Testing of pT7 37oC

Initial testing of pT7 in vitro at 37oC. This experiment will carry on until fluorescence reaches that of the control.

Protocol can be found here under Phase 1-In vitro testing on the experimental design page.

Results can here under Results on the experimental design page

Operating Range of in vitro Testing of pTet

Initial testing of operating ranges of pTet in vitro at 10oC and 45oC. The sampling of this was every 30minutes.

Protocol can be found here under Phase 2-Operating Temperature Ranges on the experimental design page.

Results can here under ... on the experimental design page


Vesicles Formation with GFP

Microscope pictures from sample 4 (2ml emulsion, centrifuge for 20min at 30x g). Left: A fluorescent object. Right: The same object under white light.
Strange structures. Left: Aggregates in the oil-lipid emulsion. Right: Air-water interface and an unexpected 'bubbly' structure in the aqueous region.

Formation of Vesicles

Using a prepared well-dessicated, well sonicated DOPC/mineral oil suspension from yesterday,

  • 2ml of suspension was taken to prepare an interface according to protocol.
  • 10x diluted GFP solution was used to prepare the emulsion; stirred gently with magnetic stirrer.
  • Special care was taken to protect the GFP solution from light at all times.

4 samples prepared:

  • Sample 1: 2ml of emulsion, no centrifuge.
  • Sample 2: Following the protocol, with 2ml suspension interface and 100μl of emulsion added.
  • Sample 3: 2ml of emulsion, centrifuge at 120x g for 10mins.
  • Sample 4: 2ml of emulsion, centrifuge at 30x g for 20mins.


Results

Light Microscopy (Magnification - 100x):

  • Emulsion: Vesicles seen, presumably monolayer.
  • Sample 1: Water-oil interface observed. Presumably the phospholipids have clumped altogether.
  • Sample 2: Vesicles seen.
  • Sample 3: Vesicles seen.
  • Sample 4: Vesicles seen.

Fluorescence Microscopy (Magnification - 100x):

  • Emulsion: GFP vesicles seen. Lots of GFP artifacts.
  • Sample 1: No fluorescent vesicles observed.
  • Sample 2: No fluorescent vesicles observed.
  • Sample 3: "Vesicles" of sizes 1-10 μm found encapsulating GFP within. Lots of GFP artifacts.
  • Sample 4: "Vesicles" of sizes 1-10 μm found encapsulating GFP within. Lots of GFP artifacts.


Preparations

No preparations were carried out.

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