"
User:University of Washington/10 July 2008
From 2008.igem.org
(→lambda red) |
|||
| (11 intermediate revisions not shown) | |||
| Line 1: | Line 1: | ||
== RP4 Conjugation == | == RP4 Conjugation == | ||
| - | '''Conjugation protocol #1'''<br\> | + | '''[[Protocols| Conjugation protocol #1]]'''<br\> |
·DH5α <br\> | ·DH5α <br\> | ||
·DH5α + S17-1 w/ RP4 <br\> | ·DH5α + S17-1 w/ RP4 <br\> | ||
·DH5α + naked RP4 DNA <br\> | ·DH5α + naked RP4 DNA <br\> | ||
·DH5α + amp resistant RFP plasmid | ·DH5α + amp resistant RFP plasmid | ||
| + | |||
== Lambda Red == | == Lambda Red == | ||
·Synthesis tetR insert for KorA and trbA replacement | ·Synthesis tetR insert for KorA and trbA replacement | ||
| + | |||
| + | |||
| + | == Lamdba Red Recombineering of RP4 (Bryan) == | ||
| + | |||
| + | Attemped to PCR amplify recombinant fragments containing selectable/counter-selectable TetR cassette. Probable source of error: wrong PCR reaction buffer or primer dilution. | ||
| + | |||
| + | == AHL Production in Yeast (Bryan) == | ||
| + | |||
| + | Third attempt at transformation of C0161 LuxI gene into Top10 failed. Previous attempts also failed using electrocompetent cells. Conclusion: C0161 is faulty part. | ||
| + | |||
== non-RP4 Conjugation == | == non-RP4 Conjugation == | ||
| - | '''Mini Prep'''<br\> | + | '''Mini Prep & [[UW Glycerol Stocks| Glycerol Stock]]'''<br\> |
·CV13(Yep13)<br\> | ·CV13(Yep13)<br\> | ||
·pDPT51 | ·pDPT51 | ||
| + | |||
| + | == LuxR from AraC and TetR == | ||
| + | |||
| + | - GFP E0240 grew, got some single colonies, stored in a fridge. | ||
| + | |||
| + | - Finished up QuikChange Mutagenesis: Dpn1 digestion. | ||
| + | |||
| + | - Did PCR purification with 30 ul EB Buffer. | ||
| + | |||
| + | - Transformed mutated AraC plasmids into XL1 Blue cells. | ||
| + | |||
| + | //Note: Electroporator 200 Ohms, 1.5 kV, with Tsy Media<br> | ||
| + | //Note: Failed to put cells on ice while bringing them over to do the electroporation. Hopefully, they will still grow. | ||
| + | *Tube 1: 45 ul XL1 Blue + 15 ul DNA#1 | ||
| + | *Tube 2: 45 ul XL1 Blue + 2 ul DNA#1 | ||
| + | *Tube 3: 45 ul XL1 Blue + 15 ul DNA#2 | ||
| + | *Tube 4: 45 ul XL1 Blue + 2 ul DNA#2 | ||
| + | *Tube 5: 45 ul XL1 Blue | ||
| + | |||
| + | - Plated 200 ul each on LB agar + Amp | ||
| + | |||
| + | |||
| + | ==LuxR from pLac== | ||
| + | |||
| + | -4 samples of I736004 transformed cells were grown to mid-log phase in M9 media, then glucose, IPTG, or glucose+IPTG were added to each sample. Flourescence of each sample was measured every 30 minutes for 1.5 hours. Samples were left to grow overnight. | ||
| + | |||
| + | == BioBrick Promoter Measurements == | ||
| + | |||
| + | - Fluorescence and OD600 data was retrieved for the promoter-construct transformants and control groups. The data was manipulated and interpreted in MATLAB. A slope comparison method and a normalized-fluorescence method for singular log-phase data points were used to determine promoter strengths. The slope and data points associated with the empty TOP10 cells were accounted for. The promoter strengths gleaned from the slope-comparison method, the normalized-fluorescence method, and the values listed in the Measurement Kit are provided, respectively, below: | ||
| + | |||
| + | -I20260 (promoter J23101): 1, 1, 1 | ||
| + | |||
| + | -I20268 (promoter J23102): 1.09, 0.97, 0.96 | ||
| + | |||
| + | -I20269 (promoter J23150): 0.19, 0.24, 0.26 | ||
| + | |||
| + | -I20270 (promoter J23151): 0.44, 0.57, 0.55 | ||
| + | |||
| + | |||
---- | ---- | ||
Back to [[Team:University_of_Washington/Notebook#Notebook]] | Back to [[Team:University_of_Washington/Notebook#Notebook]] | ||
Latest revision as of 23:44, 24 July 2008
Contents |
RP4 Conjugation
Conjugation protocol #1
·DH5α
·DH5α + S17-1 w/ RP4
·DH5α + naked RP4 DNA
·DH5α + amp resistant RFP plasmid
Lambda Red
·Synthesis tetR insert for KorA and trbA replacement
Lamdba Red Recombineering of RP4 (Bryan)
Attemped to PCR amplify recombinant fragments containing selectable/counter-selectable TetR cassette. Probable source of error: wrong PCR reaction buffer or primer dilution.
AHL Production in Yeast (Bryan)
Third attempt at transformation of C0161 LuxI gene into Top10 failed. Previous attempts also failed using electrocompetent cells. Conclusion: C0161 is faulty part.
non-RP4 Conjugation
Mini Prep & Glycerol Stock
·CV13(Yep13)
·pDPT51
LuxR from AraC and TetR
- GFP E0240 grew, got some single colonies, stored in a fridge.
- Finished up QuikChange Mutagenesis: Dpn1 digestion.
- Did PCR purification with 30 ul EB Buffer.
- Transformed mutated AraC plasmids into XL1 Blue cells.
//Note: Electroporator 200 Ohms, 1.5 kV, with Tsy Media
//Note: Failed to put cells on ice while bringing them over to do the electroporation. Hopefully, they will still grow.
- Tube 1: 45 ul XL1 Blue + 15 ul DNA#1
- Tube 2: 45 ul XL1 Blue + 2 ul DNA#1
- Tube 3: 45 ul XL1 Blue + 15 ul DNA#2
- Tube 4: 45 ul XL1 Blue + 2 ul DNA#2
- Tube 5: 45 ul XL1 Blue
- Plated 200 ul each on LB agar + Amp
LuxR from pLac
-4 samples of I736004 transformed cells were grown to mid-log phase in M9 media, then glucose, IPTG, or glucose+IPTG were added to each sample. Flourescence of each sample was measured every 30 minutes for 1.5 hours. Samples were left to grow overnight.
BioBrick Promoter Measurements
- Fluorescence and OD600 data was retrieved for the promoter-construct transformants and control groups. The data was manipulated and interpreted in MATLAB. A slope comparison method and a normalized-fluorescence method for singular log-phase data points were used to determine promoter strengths. The slope and data points associated with the empty TOP10 cells were accounted for. The promoter strengths gleaned from the slope-comparison method, the normalized-fluorescence method, and the values listed in the Measurement Kit are provided, respectively, below:
-I20260 (promoter J23101): 1, 1, 1
-I20268 (promoter J23102): 1.09, 0.97, 0.96
-I20269 (promoter J23150): 0.19, 0.24, 0.26
-I20270 (promoter J23151): 0.44, 0.57, 0.55
Back to Team:University_of_Washington/Notebook#Notebook
