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Revision as of 11:12, 15 October 2016
Notebook
Inducible Gene Switch
- Re-suspended DNA (constitutive promoters) from iGEM Distribution kit and transformed them into DH5α strain.
- Growth was found on the negative control plates so re-transformed DNA
- Inoculated of transformed cells.
Pilot Experiment
- Made stocks of our reagents Glucose Stock for 0.005 g/mL, Glucose Oxidase Stock for 0.0025 g/mL, HRP Stock for 250 μL/mL, ABTS for 0.0025 g/mL
Inducible Gene Switch
- Performed miniprep for overnight cultures
- Prepared fresh batch of DH5α chemical competent cells
- Restriction enzyme digest of constitutive promoters and control(pUC19) for validation with EcoRI and PstI
![figure 1](https://static.igem.org/mediawiki/2016/c/c9/T--Manchester--notebook_mech2_1.png)
![figure 1](https://static.igem.org/mediawiki/2016/5/5e/T--Manchester--notebook_mech2_2.png)
Pilot Experiment
- Still awaiting the arrival of the BMG spectrophotometer
Inducible Gene Switch
- Prepared DH5α chemical competent cells
- Prepared Chloramphenicol antibiotic stock
- Restreaked alcA 1 (BBa_K678001) and spispink(BBa_K1033923 pink chromoprotein)
Pilot Experiment
- We began by preparing master mix containing three reagents glucose oxidase, HRP and ABTS (table 1)
- Six different glucose concentrations were then made as depicted in table 2
- Protocol
- 50 μl of glucose solution was added into each well. Samples were run in triplicates
- Tube containing master mix was placed into spectrophotometer.
- Spectrophotometer was then used to measure absorbance of our green coloured product-oxidised ABTS at 420 nm. Absorbance values were taken every 2 sec for 3 min once 150 ul of master mix was added into each well.
- Results
- The rate of reaction did not level off and poor colour intensity was observed.
- We repeated the same experiment we did earlier this week. However instead of measuring absorbance for 3 min absorbance was measured every 2 sec for a 5 min period.
- Results - Reaction rate did level off after measuring absorbance for 5 min. However, colour intensity was still very poor.
Final reagent (μg/ml) | |
---|---|
Glucose oxidase | 60 |
HRP | 60 |
ABTS | 100 |
Final glucose concentration (μg/ml) |
---|
0.50 |
1.00 |
.25 |
1.50 |
1.75 |
2.00 |
Inducible Gene Switch
- Important event:
18th July - Resurrected DNA from iGEM DNA distribution kit - Resuspended DNA(RBS, amilCP(blue chromoprotein) and amilGFP(yellow chromoprotein)) from iGEM Distribution kit and transformed them into DH5α strain.
- Inoculated alcA 1 and spispink for miniprep
Pilot Experiment
- To increase the brightness of the colour change we repeated the experiment we performed on 22/07/2016. However this time we doubled the concentration of each reagent that made up the master mix (table 3).
- Based on results from yesterday, further increases in in reagents concentration that make up the master mix were tested. We tested the same glucose concentration as on 22/07/2016. The same parameters for the absorbance reading were used (i.e. every 2 sec for 5 min). The only parameter that was changed this time was master mix reagent concentration (table 4)
- Today it was decided to increase glucose concentration (table 5) following previous experimental conditions that did not result in appearance of intense green colour. Concentration of master mix reagents and absorbance time window was kept the same as on 22/07/2016. Samples were run in triplicates
Final reagent concentration (μg/ml) | |
---|---|
Glucose oxidase | 120 |
HRP | 20 |
ABTS | 200 |
Result
Increasing the concentration of master mix reagent did not yield intense colour changes (figure 1)
![figure 1](https://static.igem.org/mediawiki/2016/1/1f/T--Manchester--notebook_mech1_1.png)
Final reagent concentration (μg/ml) | |
---|---|
Glucose oxidase | 625 |
HRP | 62.5 |
ABTS | 625 |
Result
Strongly visible green colour was not observed following today’s experiment
Final glucose concentration (μg/ml) |
---|
0.50 |
1.00 |
.25 |
1.50 |
1.75 |
2.00 |
Result
At the end of our experiment we noticed very intense colour change as shown in figure 2.
![figure 2](https://static.igem.org/mediawiki/2016/5/54/T--Manchester--notebook_mech1_2.png)
Inducible Gene Switch
- Ordered genes from IDT: alcA2 and alcR
- Inoculated CP1 and CP3
Pilot Experiment
- Performed the following reaction where ABTS is oxidised in the presence of H2O2 (final concentration 250 μg/ml ) and HRP (final concentration 250 μg/ml
- Just measured absorbance of water at 420 nm
- Set up the reaction with all 4 reagents. Measured absorbance every 25 sec for 900 sec. Run samples in triplicates
Concentration of ABTS (μg/ml) |
---|
1 |
5 |
10 |
15 |
20 |
Concentration (μg/ml) | |
---|---|
HRP | 0.25 |
HRP | 2.50 |
H2O2 | 5.08 |
ABTS (μl) | H2O2 (μl) | HRP (μl) | PBS (μl) | |
---|---|---|---|---|
Sample 1 | 10 | 5 | 5 | 180 |
Sample 2 | 15 | 7.5 | 5 | 172.5 |
Sample 3 | 20 | 10 | 5 | 165 |
Sample 4 | 25 | 12.5 | 5 | 157.5 |
Sample 5 | 30 | 15 | 5 | 150 |
Sample 6; | 35 | 17.5 | 5 | 142.5 |
Stocks | |
---|---|
H2O2 | 0.05g/ml |
ABTS | 0.0025g/ml |
HRP | 0.00025g/ml |
Inducible Gene Switch
- Important dates
2nd August- Received requested parts from iGEM HQ
5th August- verified all parts from the DNA distribution kit - Restriction enzyme digest for ligation of CP2 into J61002 plasmid(backbone for CP1 and CP3) for rfp quantification as CP2 was originally in pSB1A2 vector which does not contain rfp.
- Gel extraction of CP2 (insert) and (vector)
Expected band size of
CP2 = 2056 bp and 58 bp
CP1 = 2925 bp and 58 bp
* CP2- extract smaller fragment
* CP1- extract larger fragment
Ran samples on 1% agarose gel with 2-log ladder. Restriction digest was successful for both CP2 and CP1. However, only CP1 was successfully extracted from gel as CP2 was lost from the running gel due to small fragment size.
Concentration of CP1 after gel extraction
- Q5 polymerase PCR for CP2 using protocol
Forward primer = V2 forward from kit
Reverse primer = VR reverse from kit
Tm = 70C - Received parts from iGEM HQ in the form of bacteria on agar. All came in pSB1C3 + CmR resistance. Re-streaked them onto CmR plates, incubate O/N at 37C to get single colonies next day.
- Overnight ligation of CP2 (insert) + CP1 (vector) with T4 ligase using protocol
- Transformation of ligated products in DH5α chemical competent cells
- Colony PCR for CP1 and CP2 transformants
- Expected colony PCR fragment size = 1142bp
- Ran colony PCR products on 1% agarose gel to check if ligation was successful. No bands were seen indicating the ligation failed.
- Restriction digest to confirm all vectors plasmids that we need from BioBrick kit in 10 ɥl reaction (1ɥl DNA).
- Ran digested products on 1% agarose gel to check with a 2-log ladder
- Ran CP2 PCR product on 4% agarose gel with 2-log DNA ladder and low MW ladder to check if amplified region was correct
![figure 7](https://static.igem.org/mediawiki/2016/9/92/T--Manchester--notebook_mech2_7.png)
![figure 8](https://static.igem.org/mediawiki/2016/4/4c/T--Manchester--notebook_mech2_8.png)
![figure 9](https://static.igem.org/mediawiki/2016/1/1e/T--Manchester--notebook_mech2_9.png)
![figure 10](https://static.igem.org/mediawiki/2016/a/ad/T--Manchester--notebook_mech2_10.png)
![figure 11](https://static.igem.org/mediawiki/2016/e/eb/T--Manchester--notebook_mech2_11.png)
![figure 12](https://static.igem.org/mediawiki/2016/8/81/T--Manchester--notebook_mech2_12.png)