Contents 1 September 1.1 September 2, 2010 1.1.1 ADS 1.2 September 20, 2010 1.2.1 ADS 1.3 September 21, 2010 1.3.1 ADS 1.4 September 21, 2010 1.4.1 ADS 1.5 September 23, 2010 1.5.1 JV 1.5.2 ADS 1.6 September 24, 2010 1.6.1 ADS 1.7 September 25, 2010 1.7.1 JV

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 -

Ingredient	Volume(µL)
MilliQ H20 Water	36.1
NEBuffer 2 (10x)	5
Upstream Part (Lumazine)	26.4
EcoRI-HF	1
SpeI	1
100X BSA	0.5

Ingredient	Volume(µL)
MilliQ H20 Water	24.6
NEBuffer 2 (10x)	5
Downstream Part (dT)	17.9
XbaI	1
PstI	1
100X BSA	0.5

Ingredient	Volume(µL)
MilliQ H20 Water	3.35
NEBuffer 2 (10x)	2.5
Destination Plasmid (pSB1C3)	17.9
EcoRI-HF	.5
PstI	.5
100X BSA	0.25

• 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 37o. Followed by 20 minute heat shock at 80o.
      • • 2(µL) was removed from the heat killed reactions for analysis on gel.

September 20, 2010

ADS

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

Lane	Sample	Volume Sample (µL)	Volume Loading Dye (µL)
1	HB1	10	2
2	HB2	10	2
3	HB3	10	2
4	HB4	10	2
5	HB5	10	2
6	100bp Ladder (Fermentas)	0.5	2(+10 H20)
7	RFP1	10	2
8	RFP2	10	2
9	RFP3	10	2

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.

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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:

Lane	Sample	Volume Sample (µL)	Volume Loading Dye (µL)
1	xylE F-S 1	5	1
2	xylE F-S 2	5	1
3	xylE F-S 3	5	1
4	xylE F-S 4	5	1
5	xylE F-S 5	5	1
6	xylE F-S 6	5	1
7	100bp Ladder (NEB)	0.5	1(+5 H20)
8	xylE S-F 1	5	1
9	xylE S-F 2	5	1
10	xylE S-F 3	5	1
11	xylE S-F 4	5	1
12	xylE S-F 5	5	1
13	xylE S-F 6	5	1
14	Empty
15	PCR of K118021-B0015 (ADS)	1	1
16	Empty
17	Empty

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

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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

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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

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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 -80^oC.