IGEM:PennState/Labbook/LucienWeiss: Difference between revisions

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#*BBa_B0010 Terminator
#*BBa_B0010 Terminator
#*BBa_B0012 Terminator
#*BBa_B0012 Terminator
#*
#Built so far and in registry:
#Built so far and in registry:
#*BBa_I14032 LacIQ constit Promoter
#*BBa_I14032 LacIQ constit Promoter
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#*=BBa_J09855
#*=BBa_J09855
#**24G iGEM 2007 Parts Kit Plate 3 pSB1A2 V1001
#**24G iGEM 2007 Parts Kit Plate 3 pSB1A2 V1001
#Cloning Plan Step 1:
#Cloning Plan Step 1:
#*Prefix Insertion BBa_J52008 Rluc Luciferase before BBa_B0015 (BBa_B0010 Terminator + BBa_B0012 Terminator)
#*Prefix Insertion BBa_J52008 Rluc Luciferase before BBa_B0015 (BBa_B0010 Terminator + BBa_B0012 Terminator)

Revision as of 10:55, 8 June 2007

Lucien Weiss Lab Notebook


Experiment Aims

Test motility on a variety of surface hardnesses in order to determine optimal level to use for assays.

Procedure

Eiken Agar Plates were made using a modified version of general procedure 1. Increased agar concentrations were made to increase hardness of gel and reduce the speed of motility.

.20% Eiken Agar Plates:

  • .25g Bactotryptone
  • .20g NaCl
  • .15g sodium citrate
  • .05g Eiken Agar (for 20EikenAgar)

.30% Eiken Agar Plates:

  • .25g Bactotryptone
  • .20g NaCl
  • .15g sodium citrate
  • .075g Eiken Agar (for 30EikenAgar)

.40% Eiken Agar Plates:

  • .25g Bactotryptone
  • .20g NaCl
  • .15g sodium citrate
  • .10g Eiken Agar (for 40EikenAgar


  1. Constructs were transformed into RP3087
    • YFP Sender Cells-Sender (LVA+)-RP3087 w/ [I14032+B0030+C0161 A3]+E0430
    • Sender Cells-Sender-RP3087 w/ I14032+B0030+C0161 (B2)
    • Receiver-RFP+Receiver-RP3087 w/ I13321+[J09855+B0033+J09274]
  2. Grown overnight at 37 degrees C on 50 micromolar Kan LB plates
  3. 3mL cultures inoculated off of the plate and grown at 30 degrees C until OD590~.45
  4. 3uL placed of receiver cells placed in various distances from 3uL inoculated sender colony.

Results:

.20% Eiken Agar Sender and Receivers
.30% Eiken Agar with sender and receiver cells after 8 hours
.30% Eiken Agar Sender and Receivers
.30% Eiken Agar with sender and receiver cells after 8 hours
.30% Eiken Agar with regular sender and receiver cells after 20 hours
.30% Eiken Agar with Degradation Tagged AHL
.30% Eiken Agar with sender produced AHL tagged for degradation and receiver cells after 8 hours
.30% Eiken Agar with sender cells producing AHL tagged for degradation and receiver cells after 20 hours
.40% Eiken Agar Sender and Receivers
.40% Eiken Agar with sender produced AHL tagged for degradation after 8 hours
.40% Eiken Agar with regular sender and receiver cells after 8 hours


Observations:

.20% agar plate difficult to handle as agar was extremely fragile. Results were diminished by heavy diffusion. .30% agar results in fair motility and is much easier to handle .40% agar results in no motility

Conclusions:


Future Experiments:

Retry .20% Agar experiments with set distances away from sender Experiment with temperature changes

March 13, 2007: Motility Assay

Experiment Aims

Test induced motility at a variety of distances from sender cells.

Procedure

Eiken Agar Plates were made using a modified version of general procedure 1. Agar concentrations were kept at .20%

.20% Eiken Agar Plates:

  • .25g Bactotryptone
  • .20g NaCl
  • .15g sodium citrate
  • .05g Eiken Agar (for 20EikenAgar)


  1. Constructs were transformed into RP3087
    • YFP Sender Cells-Sender (LVA+)-RP3087 w/ [I14032+B0030+C0161 A3]+E0430
    • Sender Cells-Sender-RP3087 w/ I14032+B0030+C0161 (B2)
    • Receiver-RFP+Receiver-RP3087 w/ I13321+[J09855+B0033+J09274]
  2. Grown overnight at 37 degrees C on 50 micromolar Kan LB plates
  3. 3mL cultures inoculated off of the plate and grown at 30 degrees C until OD590~.45
  4. 3uL placed of receiver cells placed in various distances from 3uL inoculated sender colony.

Results:

.20% Eiken Agar Sender and Receivers
.20% Eiken Agar with sender and receiver cells after 8 hours
.20% Eiken Agar with sender and receiver cells after 8 hours


Observations:

Difficult to discern motility on .20% Eiken Agar plates

Conclusions:


Future Experiments: Increase Agar Concentration Experiment with temperature changes

March 16, 2007: Motility Assay

March 29-April 1, 2007: Institute of Biological Engineering

Poster: Bacterial Relay Race
IBE Poster

April 22, 2007: Project Proposal Presentations

<table|left>

Project Proposal: Pressure Sensing in Bacteria

Slide1
Slide2
Slide3
Slide4
Slide5
Slide6
Slide7
Slide8

<table|left>

Project Proposal: Bi and Sept Stable Oscillator

Slide1
Slide2
Slide3
Slide4
Slide5
Slide6
Slide7

April 28, 2007: Synthetic Biology 3.0

Biology 3.0 Website

May 15, 2007: General Procedures

These are all the general cloning kinds of things that will be used to build our constructs. We will probably get into getting parts sequenced if things do not seem to be working, but that gets expensive and takes a couple of days, so my thoughts are we should save that until it looks like we are getting into trouble. Labeling is key.

Labeling

  1. First note this is the most important thing for how easy it is. In the past we have had a lot of problems however so I have come up with a system. Remember everyone is going to need to be able to read this, so use a fine tipped sharpie always. Also EtOH is an organic solvent and will remove your title. Be careful with this especially during minipreps. Write on the outside of the tube, Thats really obvious it would seem, but just a warning.

Microcentrifuge tubes On Cap:

Row 1: Initials Date
Row 2: Part/parts (this is a quick reference, always know what it is that you are using)
Row 3: ID number 0001-9999 (each member will have a list of every tube they have online)

On Side: you may want to duplicate all the information especiall if it is very messy or you needed more room.

EX-on Cap:

LW 5-19-07
B33+J2971
0012

On Side:

LW 5-19-07
B0033+J02971 miniprep
Row 3: 0012

Innoculation from plate

  1. flame loop
  2. cool loop on plate
  3. pick up individual colony
  4. place in culture and shake loop
  5. flame loop

Culture Dilutions EX 1-1:100 Dilution

  1. Add 3mL fresh LB to a new culture tube
  2. Add 3uL of appropriate (1:1000)antibiotic solution
  3. Take 30uL from starter culture and place in fresh media.
    • Becareful to not touch the sides of the tube with the pipetmen.

Checking cell OD. Using Spectrophotometer

  1. Turn on spectrophotometer (switch on the back to the right)
  2. Allow the startup to run, and then turn off deuterium Light (needed at hv <300nm)
  3. Turn wavelength to 590nm
  4. Zero with 1mL LB in Cuvette
  5. Do an appropriate dilution (suggested 1:5, 1:10) of culture with fresh LB into a fresh Cuvette
    • note 1: if you are trying to find out how long it will be until cells are at OD .6 (mid log) you can take the OD at two seperate times and use http://molbiol.edu.ru/eng/scripts/01_02.html.
    • note 2: diluting a culture down to OD .6 will not put it in mid log phase; dilute down to .15 and allow it to grow back up to .6 if your culture has grown too long.

note: wavelength for DNA = 260nm, wavelength for protein=280nm, to find purity take ratio between peaks.

Miniprep

  1. Spin down 3mL of saturated cell culture, remove supernatant
  2. Resuspend pelleted bacteria in 250uL Buffer P1 and transfer to a 1.5mL micro centrifuge tube.
  3. Add 250uL Buffer P2 and invert the tube gently 4-6 times to mix.
  4. Add 350uL Buffer N3 and invert the tube immediately but gently 4-6 times.
    • the solution should be come cloudy
  5. Centrifuge for 10 minutes at 13,000 rpm in a table-top microcentrifuge
    • allign tubes such that the cap clip is facing outward
  6. decant supernatant into spin column
  7. Centrifuge for 60s. Discard flow-through.
  8. Add .75mL Buffer PE and centifuge for 60s.
  9. Discard flowthrough and centrifuge for 60s
  10. Place the spin column into a new 1.5mL microcentrifuge tube.
  11. Add 30-50uL of water (8.5>pH>7) to the center of each spin column.
  12. Let tubes stand vertically for 1min, then centrifuge for 1min.

Digest

  1. Add components generally by largest volume to smallest. I tend to add water first just so I do not waste pipettips if I am using different vectors, but cetainly do not start with .5 or 1 uL components.
  2. Choosing your Enzymes: The biobricks are built with the restriction sites as following: -EcoR1-Xba1-Part-Spe1-Pst1- it is important to remember that your plasmid may have some of these restriction sites too, so if too many bands are appearing on the gel becareful of this. Keep these enzymes on ice when they are out of the feezer, and put them back immediately after you use them. If enzymes happen to be left out please let me know and we will decide what to do with them. Do not put enzymes that have been left out back in the freezer, or future restrictions will not work.
  3. Choosing your buffer: Check the compatibility with each enzyme and the buffer. There should be a list for double digestions.
  4. Forgetting to add BSA will make the whole thing not work.
  • Examples demonstrate the two main digets that will be used in this lab.

EX1-10uL Digest

  1. 3.0uL H2O
  2. 4.0uL Vector
  3. 1.0uL Buffer X
  4. 1.0uL BSA
  5. 0.5uL Enzyme 1
  6. 0.5uL Enzyme 2

EX2-20uL Digest

  1. 4.0uL H2O
  2. 11.uL Vector
  3. 2.0uL Buffer X
  4. 2.0uL BSA
  5. 0.5uL Enzyme 1
  6. 0.5uL Enzyme 2

Running a Gel TAE Buffer-if you are extracting from your gel TBE Buffer-for better imaging Do not load DNA in high concentrations. It may be tempting to put in as much concentrated DNA as you can, but this is not a good thing.

Gel Extraction


Ligation

Transformation

May 21, 2007: Summer Schedule and Restriction Enzymes

<table|left>

Summer Schedule and Week's Plan

Slide1
Slide2
Slide3
Slide4
Slide5
Slide6
Slide7
Slide8
Slide9

Restriction Enzymes

|---------EcoRI--------| |---------XbaI---------| |---------PART---------| |---------SpeI---------| |---------PstI---------|
5' G|AATT C 3' 5' T|CTAG A 3' 5' A|CTAG T 3' 5' C TGCA|G 3'
3' C TTAA|G 5' 3' A GATC|T 5' 3' T GATC|A 5' 3' G|ACGT C 5'

May 22, 2007: Growing Parts From Registry Plates

Registry plates are kept in the -4 degree freezer in a box labeled registry plates.

  1. Take a 10uL pipetment and pipet 10uL of dd-water up and down inside target well on the registry plate
  2. Once the water appears red place that in a labeled microcentrifuge tube (Initials Date, Part name well #, and ID number).
    • Now the registry part is ready to be transformed. Proceed with regular transformation.

May 24, 2007: Gel Results: J32038 & J31004 Digests

Gel 1 Results: J32038 & J31004 Digests Digests in Noah Johnsons Labnotebook 5-24-07 Restriction Digest DNA

EcoRI+SpeI J31004 A EcoRI+SpeI J31004 B EcoRI+SpeI J31004 C EcoRI+XbaI J32038 A EcoRI+XbaI J32038 B EcoRI+XbaI J32038 C EcoRI J31004 A XbaI J31004 A SpeI J31004 A J31004 A
LW0003 LW0004 LW0005 LW0006 LW0007 LW0008 LW0003 LW0003 LW0003 LW0003
No bands No bands No bands No bands 4 bands 2 bands 1 band 1 band 1 band no bands
Gel Leaked Gel Leaked Gel Leaked Gel Leaked Incomp Digestion Success Success Success Success Gel Leaked
Wrong Buff Wrong Buff Wrong Buff
600bp Extracted 3kbp Extracted 3kbp Extracted

Gel 2 Results: J32038 & J31004 Digests Digests in Noah Johnsons Labnotebook 5-24-07 Restriction Digest

EcoRI+XbaI J32038 A EcoRI+XbaI J32038 B EcoRI+XbaI J32038 C EcoRI+SpeI J31004 A EcoRI+SpeI J31004 B EcoRI+SpeI J31004 C J32038 C J31004 C
LW0006 LW0007 LW0008 LW0003 LW0004 LW0005 LW0008 LW0005
1 band 1 band 1 band 2 bands 2 bands 2 bands 4 bands 4 bands
Success Success Success Success Success Success Success Success
3kbp Extracted 3kbp Extracted 3kbp Extracted 600bp Extracted 600bp Extracted 600bp Extracted

May 30, 2007: Bacterial Relay Race Plan

<table|left>

Bacterial Relay Race

Slide1
Slide2
Slide3
Slide4
Slide5
Slide6

June 08, 2007: Bacterial Relay Race Plan

  1. Receiver Cells:
    • BBa_I14032 LacIQ constit Promoter
    • BBa_B0034 RBS
    • BBa_C0062 LuxR (to bind to HSL)
    • BBa_B0010 Terminator
    • BBa_B0012 Terminator
    • BBa_R0062 PluxR HSL+LuxR activates
    • BBa_B0033 Weak RBS
    • BBa_J09271 motB without RBS
    • BBa_J52008 Rluc Luciferase
    • BBa_B0010 Terminator
    • BBa_B0012 Terminator
  2. Built so far and in registry:
    • BBa_I14032 LacIQ constit Promoter
    • BBa_B0034 RBS
    • BBa_C0062 LuxR (to bind to HSL)
    • BBa_B0010 Terminator
    • BBa_B0012 Terminator
    • BBa_R0062 PluxR HSL+LuxR activates
    • =BBa_J09855
      • 24G iGEM 2007 Parts Kit Plate 3 pSB1A2 V1001
  1. Cloning Plan Step 1:
    • Prefix Insertion BBa_J52008 Rluc Luciferase before BBa_B0015 (BBa_B0010 Terminator + BBa_B0012 Terminator)
      • BBa_J52008 12E iGEM 2007 Parts Kit Plate 3 pSB1AK3 V1001
      • BBa_B0015 1I iGEM 2007 Parts Kit Plate 1 pSB1AK3 V1001
      • BBa_B0015 3O iGEM 2007 Parts Kit Plate 3 pSB1AK3 V1010
    • Suffix Insertion BBa_J09271 motB without RBS after BBa_B0033 Weak RBS
      • 24G iGEM 2007 Parts Kit Plate 3 pSB1A2 V1001
      • 3M iGEM 2007 Parts Kit Plate 1 pSB1A2 V1004
  2. Cloning Plan Step 2:
    • Prefix Insertion BBa_J09271+BBa_B0033 before BBa_J52008+BBa+B0010+BBa_B0012
    • Suffix Insertion BBa_J52008+BBa+B0010+BBa_B0012 after BBa_J09271+BBa_B0033
    • (use whichever works)
  3. Cloning Plan Step 3:
    • Suffix Insertion: BBa_J09271+BBa_B0033+BBa_J52008+BBa+B0010+BBa_B0012 after BBa_J09855
    • Prefix Insertion: BBa_J09855 before BBa_J09271+BBa_B0033+BBa_J52008+BBa+B0010+BBa_B0012
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