Team:Lethbridge/Notebook/Lab Work/September

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<b>Objective:</b> PCR xylE to attach fusion standard to the N-term and standard suffix to C-term.<br>
<b>Objective:</b> PCR xylE to attach fusion standard to the N-term and standard suffix to C-term.<br>
 +
 +
<table border ="3">
 +
<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x6)(&micro;L)</b>
 +
<tr><td>Milli-Q H<sub>2</sub>O<td>12.8<td>76.8
 +
<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>12
 +
<tr><td>dNTPs<td>1<td>6
 +
<tr><td>Fusion Forward Primer<td>1<td>6
 +
<tr><td>Standard ReversePrimer<td>1<td>6
 +
<tr><td>Template DNA<td>2<td>12
 +
<tr><td>Pfu polymerase<td>0.2<td>1.2
 +
</table><br>
 +
 +
Fusion standard prefix has melting temperature of 66.5<sup>o</sup>C.  Standard suffix has melting temperature of 70.5<sup>o</sup>C. 
 +
Annealing temperature of 61.5<sup>o</sup>C with 5 <sup>o</sup>C gradient. 
 +
PCR- Conditions:
 +
1.  95<sup>o</sup>C for 180 sec
 +
2.  95<sup>o</sup>C for 30 sec
 +
3.  61.5 +/- 5<sup>o</sup>C for 30 sec
 +
4.  72<sup>o</sup>C for 150 sec
 +
5.  72<sup>o</sup>C for 600 sec
 +
(35 cycles)
==<font color="white">September 18, 2010==
==<font color="white">September 18, 2010==

Revision as of 17:18, 23 October 2010



Feel free to look around our notebook!

Here you can check out the work we have done in the lab, click on a month to take a look!

Contents

September

September 2, 2010

ADS

Objective: Assemble lumazine-dT into pSB1C3 using NEB BioBrick Assembly Kit.
Method: Have the following parts: dT [28 ng/(µL)]; lumazine [78 ng/(µL)]; psB1C3 [14 ng/(µL)].

  • Require 500 ng DNA each part in a 50(µL) rxn therefore 17.9(µL) dT and 6.4(µL) Lumazine. Need 250 ng pSB1C3 or 17.9(µL)

Restriction Reactions -

IngredientVolume(µL)
MilliQ H20 Water36.1
NEBuffer 2 (10x)5
Upstream Part (Lumazine)26.4
EcoRI-HF1
SpeI1
100X BSA0.5


IngredientVolume(µL)
MilliQ H20 Water24.6
NEBuffer 2 (10x)5
Downstream Part (dT)17.9
XbaI1
PstI1
100X BSA0.5


IngredientVolume(µL)
MilliQ H20 Water3.35
NEBuffer 2 (10x)2.5
Destination Plasmid (pSB1C3)17.9
EcoRI-HF.5
PstI.5
100X BSA0.25


  • 2(µL) was removed prior to adding enzymes for analysis on gel.
    • Split digestion reactions in half: dT - 2 @ 24(µL); Lum - 2 @ 24(µL); psB1C3 - 2 @ 11.5(µL)
      • Ran 10 minute and 60 minute reactions at 37oC. Followed by 20 minute heat shock at 80oC.
        • 2(µL) was removed from the heat killed reactions for analysis on gel.
  • Performed 4 combinations of ligation: 10 min Restriction + 10 min Ligation; 10 min Restriction + Overnight Ligation; 60 min Restriction + 10 min Ligation; 60 min Restriction + Overnight Ligation


Ingredient1X(µL)2X Master Mix(x2.5)(µL)
MilliQ H20 Water1177.5
T4 Ligase Buffer (10x)25
Upstream Part25
Downstream Part 25
T4 DNA Ligase12.5
Plasmid Part25

Incubated 10 minute and overnight ligations at room temperature ( 25oC). Heat killed ligase at 80oC for 20 min.

  • 2(µL) of each reaction was taken for analysis on gel.
  • Confirmed ligation with PCR analysis. Analyzed restriction, ligation and PCR on a 1.5% TAE agarose gel.
Component1X(µL)Master Mix(x5.5)(µL)
Milli-Q H2O36.5202.4
10x Pfu Buffer with MgSO4527.5
dNTPs211
Forward VF2 Primer211
Reverse VR Primer211
Template DNA2
Pfu polymerase0.21.1

Added 48(µL) to each reaction.

GEL PICTURE!!!

Objective: Characterize xylE in LB Media vs. M9 Minimal Media.
Method: Three experiments with spectra.

  • Experiment 1 - Measure absorption spectra of 1:10 dilution of: 1. Cells suspended in m9 media +/- catechol. 2. Cells suspended in LB media +/- catechol. 3. m9 media from spun down cells +/- catechol. 4. LB media from spun down cells +/- catechol. 5. Sterile LB media +/- catechol. 6. Water +/- catechol.
  • Experiment 2 - Catechol Breakdown. Follow absorbance of 2-hydroxymucanate semialdehyde at 375 nm. Measured all the above samples for 10 min at that wavelength.

September 14, 2010

JF

Objective: Miniprep of RFP expression construct (J04450) via Qiaquick/Qiaprep spin column. Protocol followed as per kit instructions.

JV

Objective: PCR the pSB1C3 plasmid from part J04450 miniprep.

Component1X(µL)
Milli-Q H2O12.8
10x Pfu Buffer with MgSO42
dNTPs1
SB-prep-26 Primer1
SB-prep-3P Primer1
Template DNA2
Pfu polymerase0.2

Used iGEM cycle PSB1C3 in thermocycler. Ran PCR product on 1% TAE agarose gel.

KG

Objective: PCR of xylE (mRBS-xylE K118021) with fusion standard so that an arginine tail can be attached.

Component1X(µL)Master Mix(x6)(µL)
Milli-Q H2O12.876.8
10x Pfu Buffer with MgSO4212
dNTPs16
xylE Fusion Prefix Forward Primer16
xylE Fusion Suffix Reverse Primer16
Template DNA212
Pfu polymerase0.21.2

PCR- Conditions: 1. 95oC for 180 sec 2. 95oC for 30 sec 3. 56.6 +/- 5oC for 30 sec 4. 72oC for 150 sec 5. 72oC for 600 sec (35 cycles)

Analyzed PCR on 2% agarose gel which ran at 100 V for 90 minutes.

AS

Objective: Assemble xylE-dT using 3AB assembly and 3AB assembly.

Restriction Reactions -

3AB Upstream Part (xylE)

IngredientVolume(µL)
MilliQ H20 Water30.6
NEBuffer 2 (10x)5
Plasmid DNA 11.9
EcoRI-HF1
SpeI1
100X BSA0.5

3AB Downstream Part (dT)

IngredientVolume(µL)
MilliQ H20 Water24.6
NEBuffer 2 (10x)5
Plasmid DNA17.9
XbaI1
PstI1
100X BSA0.5

3AB Plasmid (pSB1C3)

IngredientVolume(µL)
MilliQ H20 Water
NEBuffer 2 (10x)5
Plasmid DNA42.5
EcoRI-HF1
PstI1
100X BSA0.5
  • Ran 10 minute and 60 minute reactions at 37oC. Followed by 20 minute heat shock at 80oC.

Ligation Reaction -

Ingredient1X(µL)
MilliQ H20 Water11
T4 Ligase Buffer (10x)2
Upstream Part2
Downstream Part 2
T4 DNA Ligase1
Plasmid Part2

Incubated 10 minute and overnight ligations at room temperature ( 25oC). Heat killed ligase at 80oC for 20 min.

Transformation -

A) Thawed 200(µL) Library Efficiency DH5alpha Competent Cells on ice. B) Gently mixed cells and then aliquoted 100(µL) into chilled polypropylene tubes. C) Added 1(µL) of ligation mix to cells. Added 5(µL) of pUC19 DNA to 100(µL) cells to determine efficiency. D) Incubated cells on ice for 30 minutes. E) Heat shocked cells for 45 seconds in a 42oC water bath. F) Placed on ice for 2 minutes. G) Added 0.9 mL of room temperature SOC medium. H) Shook at 225 rpm for 1 hour. I) Diluted control cells 1:100 with SOC medium. J) Spread 100(µL) of this dilution on LB-Amp agar plates K) Spread 50 and 250(µL) of experimental cells on LB-Cam agar plates. L) Incubated overnight at 37oC

September 15, 2010

JV, ADS

Objective: Determine if dT ligated onto p-rbs-xylE using PCR analysis.

Component1X(µL)Master Mix(x4.5)(µL)
Milli-Q H2O11.853.1
10x Pfu Buffer with MgSO429
dNTPs14.5
Forward VF2 Primer14.5
Reverse VR Primer14.5
Template DNA1
Pfu polymerase0.2

Used iGEM program 7 in thermocycler.

Objective: Determine if minipreps from Sept 15, 2010 worked. Analyze assembly intermediates.
Method: Run 1% TAE agarose gels at 100 V for 90 minutes.

GEL PICTURES!!!

September 16, 2010

JV

Objective: Isolate RFP in pSB1C3 from DH5alpha cells.
Method: Used Qiagen minipep protocol and spin column.
Results: Gel of miniprepped RFP against previously prepared RFP showed a band of the right size.

GEL PICTURE!

September 17, 2010

JV

Objective: Isolate plasmid DNA from DH5alpha cells: K249024; B0015; K118021/B0015
Method: Used Qiagen minipep protocol and spin column. Ran a 1% agarose gel to view DNA

GEL PICTURE!

AV

Objective: PCR out RFP from the complete construct (J04450) with a N-term fusion standard.

Composition of each PCR tube:

ComponentVolume(µL)
10x Pfu Buffer w/ MgSO42
dNTP (10mM)1
N-term Fus Prefix1
Biobrick Suffix1
MilliQ H2O12.8
Template DNA (J04450)2
Pfu DNA Polymerase0.2

KG

Objective: PCR xylE to attach fusion standard to the N-term and standard suffix to C-term.

Component1X(µL)Master Mix(x6)(µL)
Milli-Q H2O12.876.8
10x Pfu Buffer with MgSO4212
dNTPs16
Fusion Forward Primer16
Standard ReversePrimer16
Template DNA212
Pfu polymerase0.21.2

Fusion standard prefix has melting temperature of 66.5oC. Standard suffix has melting temperature of 70.5oC. Annealing temperature of 61.5oC with 5 oC gradient. PCR- Conditions: 1. 95oC for 180 sec 2. 95oC for 30 sec 3. 61.5 +/- 5oC for 30 sec 4. 72oC for 150 sec 5. 72oC for 600 sec (35 cycles)

September 18, 2010

JS, DM

Objective: Overexpress mms6.
Method: Followed overexpression protocol. One flask was induced with 250(µL) IPTG while another was induced with 50(µL).

Time (minutes)OD #1 (600λ)OD #2 (600λ)
300.1390.133
600.2000.188
900.3720.360
1200.7020.704
150(T0)0.8710.810
210(T1)2.6002.500
270(T2)3.3173.300
330(T3)4.4083.946

  • Plated cells in presence of IPTG, Fe or IPTG and Fe on AMP(+) plates. Incubated at 37 oC.
    • Preparation of these samples was achieved by taking a 60(µL) cell sample and inoculating 5 mL LB media. These solutions were incubated at 37 oC for 30 min. To 1 mL of culture, the appropriate amount of Fe2+ [0.938(µL)], Fe3+[1.88(µL)] or IPTG [1(µL)] was added.

September 20, 2010

ADS

Objective: Analyze PCR of mms6 (HB) and fluorescent proteins (AV).
Method: Analyzed on 2% TAE agarose gel.
Load order:

LaneSampleVolume
Sample (µL)		Volume Loading
Dye (µL)
1HB1102
2HB2102
3HB3102
4HB4102
5HB5102
6100bp Ladder (Fermentas)0.52(+10 H20)
7RFP1102
8RFP2102
9RFP3102

Results: ADD IMAGE

  • mms6 was not amplified
  • RFP was amplified
    • Expected size is ~800bp
    • Actual size is >1000bp
      • We believe that the suffix segment of the fusion primer annealed rather than the FP segment. This would cause the promoter (R0040) of J04450 to be amplified, adding ~200bp, giving a final size of >1000bp.

Objective: Confirm assembly (3 antibiotic) of K118021 and B0015 from Sept. XX, 2010 via PCR.
Method: Set up 50uL reaction mixture with 2uL of ligated DNA. Used VF2 and VR primers.
Used PFU setting on Thermocycler
Results: ADD IMAGE
Conclusion: Assembly did not work as intended.

September 21, 2010

ADS

Objective: Insert xylE (with N and C terminal fusion standards, obtained by PCR of K118021 by KG) into pSB1C3 plasmid for submission to registry.

Method:

  • Restriction of xylE PCR product and pSB1C3 (containing J04450 biobrick) via BioBrick Method using EcoRI-HF and PstI (Enzymes from NEB)
  • Ligation of xylE PCR product and pSB1C3 via BioBrick Method using T4 DNA ligase (Enzyme from NEB)
  • Transform into Library Efficiency Compentent DH5α Cells (Invitrogen)

Results:

  • Obtained too numerous to count (TNTC) colonies.
    • Obtained ~100 white colonies (indicating removal of RFP and insertion of new BioBrick)

Note:
Parent plasmid (from PCR) not digested, possibly K118021 moved into pSB1C3 backbone.

Follow-up:
Screen white colonies by addition of catechol to solution containing white cells.

September 21, 2010

ADS

Objective: Analyze PCR of K118021-B0015 and subsequent PCR (ADS) and PCR of <partinfo>K118021</partinfo> to add either N or C terminal fusion standard (KG).
Method: Analyze on 2% TAE agarose gel.
Load Order:

LaneSampleVolume
Sample (µL)		Volume Loading
Dye (µL)
1xylE F-S 151
2xylE F-S 251
3xylE F-S 351
4xylE F-S 451
5xylE F-S 551
6xylE F-S 651
7100bp Ladder (NEB)0.51(+5 H20)
8xylE S-F 151
9xylE S-F 251
10xylE S-F 351
11xylE S-F 451
12xylE S-F 551
13xylE S-F 651
14Empty
15PCR of K118021-B0015 (ADS)11
16Empty
17Empty

Results: ADD IMAGE

  • Both KG PCR (Standard-Fusion and Fusion-Standard) amplified
  • ADS PCR Amplified, but no insert present.

September 23, 2010

JV

Objective: Characterized catechol degradation by xylE enzyme

Method: Measured absorbance of catechol (275nm) and 2-hydroxymuconate semialdehyde (380nm).

  • Protocol:
  • 1) Grow cells in M9 minimal medium
  • 2) Take 1/10 dilution of cells
  • 3) Introduce 1µL of 0.05M catechol solution into the cell dilution. (Final concentration of 50&microM;).
  • 4) Quench the reaction with 5A% w/v trichloroacetate at certain time points. (0,15sec, 30sec, 45sec, 60sec, 2min, 3min, 4min, 5min, 10min).
  • 5) Spin down cells.
  • 6) Measure absorbance of supernatant.

Results: Cuvette used interfered with Spectra.

ADS

NOTE: In all transformations, heat shock step was missed. HOWEVER, all transformations showed significant number of colony forming units.
Objective: Move xylE (two biobrick; one with Fusion prefix, one with fusion suffix) into pSB1C3.
Method:

  • Restriction of xylE PCR product and pSB1C3 (containing J04450 biobrick) via BioBrick Method using EcoRI-HF and PstI (Enzymes from NEB)
  • Ligation of xylE PCR product and pSB1C3 via BioBrick Method using T4 DNA ligase (Enzyme from NEB)
    • Incubated 30 min at RT
  • Transform into Subcloning Efficiency Compentent DH5α Cells (Invitrogen)

Results: TBD
Follow-up: TBD

Objective: Create glycerol stocks of <partinfo>J04450</partinfo> in pSB1A3 and pSB1T3 for use in RFP-BioBrick Assembly.
Method: Transform into Subcloning Efficiency Competent DH5α Cells (Invitrogen)
Obtained all plasmid DNA from 2010 Kit Plate 1

  • J04450 in pSB1A3 - Well 1C
  • J04450 in pSB1T3 - Well 7A

Results: Obtained TNTC colonies
Follow-up:

  • Grow overnight cultures
  • Generate Glycerol Stocks
  • Generate Plasmid DNA via Maxiprep

Objective: Create glycerol stocks of received synthesized (Mr. Gene) signal peptides.
Method: Transform into Subcloning Efficiency Competent DH5α Cells (Invitrogen) plasmid DNA containing the following BioBricks:

  • 1) <partinfo>K331007</partinfo> - β-lactamase Bla Signal Sequence
  • 2) <partinfo>K331008</partinfo> - Outer Membrane Protein ompA
  • 3) <partinfo>K331009</partinfo> - Heat Stable Toxin I
  • 4) <partinfo>K331012</partinfo> - Penicillin Binding Protein DacA
  • All inserts in pMA-T vector (Standard Mr. Gene vector)

Results: Obtained TNTC Cells
Follow-up:

  • 1) Grow overnight cultures
  • 2) Purify pDNA
  • 3) Move into pSB1C3 plasmid
  • 4) Verify sequence
  • 5) Submit to registry for sequencing

September 24, 2010

ADS

Objective: Generate plasmid DNA of <partinfo>E1010</partinfo> for downstream PCR
Method: Transform plasmid DNA into Subcloning Efficiency Competent DH5α Cells (Invitrogen)
DNA obtained from 2010 Kit Plate 1 Well 18F (E1010 in pSB2K3)
Results: Obtained TBD colonies
Follow-up:

  • Grow overnight cultures (Generate glycerol stocks)
  • Purify plasmid DNA (Generate pDNA stocks)
  • PCR to add terminal fusion standards

Objective: Create glycerol stocks of J04450 in pSB1K3 for use in RFP-BioBrick Assembly.
Method: Transform into Subcloning Efficiency Competent DH5α Cells (Invitrogen)
Obtained plasmid DNA from 2010 Kit Plate 1 well 5A (J04450 in pSB1K3)
Results: Obtained TNTC colonies
Follow-up:

  • Grow overnight cultures
  • Generate Glycerol Stocks
  • Generate Plasmid DNA via Maxiprep

September 25, 2010

JV

Objective: Extract Plasmid DNA from DH5α cells.

Method:Qiagen spin column protocol.

  • <partinfo>K331007</partinfo> (in pMA-T vector)
  • <partinfo>K331008</partinfo> (in pMA-T vector)
  • <partinfo>K331009</partinfo> (in pMA-T vector)
  • <partinfo>K331012</partinfo> (in pMA-T vector)

Cells containing plasmids were put into glycerol stocks and put into HJ's -80oC.

ADS

Objective: Move plasmid DNA made by JV (above, Sept 25, 2010) into pSB1C3 for submission to the Standard Registry of Parts.
Method:

  • 1) Digest at 37o for 10 min with PstI and EcoRI:
    • pSB1C3 containing <partinfo>J04450</partinfo>
    • pMA-T with <partinfo>K331007</partinfo>, <partinfo>K331008</partinfo>, <partinfo>K331009</partinfo>, and <partinfo>K331012</partinfo>.
  • 2) Heat Kill at 80oC for 20 min
  • 3) Mix 2µL of pSB1C3 with each biobrick, ligate with T4 DNA ligase for 10 min
  • 4) Transform into subcloning efficiency DH5α cells, plate on ampicillin plates.

Results: Obtained RESULT!!! positive colonies

Objective: Assemble KG's C-terminal fusion xylE (<partinfo>K331021</partinfo>) (UNCONFIRMED) and C-terminal Arginine + dT (<partinfo>S04261</partinfo>) to create <partinfo>K331011</partinfo>.
Method:

  • 1) Digest at 37o for 10 min:
    • pSB1C3 containing <partinfo>J04450</partinfo> with PstI and EcoRI
    • <partinfo>K331021</partinfo> with EcoRI, SpeI
    • <partinfo>S04261</partinfo> with XbaI and PstI
  • 2) Heat Kill at 80oC for 20 min
  • 3) Mix 2µL of pSB1C3 with each biobrick, ligate with T4 DNA ligase for 10 min
  • 4) Transform into subcloning efficiency DH5α cells, plate on ampicillin plates.

Results: Obtained RESULT!!! positive colonies

September 26, 2010

ADS

Objective: Purify plasmid DNA from 5mL cultures grown from transformations of KG's ligated PCRs containing (UNCONFIRMED) <partinfo>K331020</partinfo> and <partinfo>K331021</partinfo>.
Method: Used Qiagen spin column method with BioBasic EZ-10 spin columns.

Objective: Assemble the following:

  • <partinfo>K331020</partinfo> + <partinfo>B0015</partinfo> to obtain To be named biobrick
  • <partinfo>K331021</partinfo> + <partinfo>S04261</partinfo> to obtain To be named biobrick

Method:

  • Digest <partinfo>K331020</partinfo> and <partinfo>K331021</partinfo> with EcoRI and SpeI
  • Digest <partinfo>B0015</partinfo> and <partinfo>S04261</partinfo> with XbaI and PstI
  • Digest <partinfo>J04450</partinfo> in pSB1C3 with EcoRI and PstI

Incubate each at 37oC for 10 min
Heat kill at 80oC for 20 min
Ligate with T4 Ligase for 10 min at room temperature
Transform into DH5&alpha subcloning efficiency cells
Results: Obtained RESULTS!!!! positive colonies.
Also analyzed minipreps on 1.5% TAE Agarose Gel
GEL!!!

September 29, 2010

ADS

Received synthesized parts from Mr. GENE (all in pMA vectors)

  • <partinfo>K331022</partinfo>
  • <partinfo>K331023</partinfo>
  • <partinfo>K331024</partinfo>
  • <partinfo>K331025</partinfo>

Each tube contained 5µg pDNA; reconstitute in 50µL of TE buffer to get 100ng/µL concentration
Transform 1µL into DH5α subcloning efficiency cells
Obtained TNTC colonies
Started overnight 5mL cultures for miniprep and insertion into pSB1C3 vector.

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