Revision as of 14:58, 15 October 2016

Manchester iGEM 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 solution 0.005 g/mL, glucose oxidase (GOx) solution for 0.0025 g/mL, horseradish peroxidase (HRP) solution 250 μL/mL, 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (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

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: GOx, HRP and ABTS (table 1). PBS was used as a solvent.

	Final reagent concentration, (μg/ml)
GOx	60
HRP	60
ABTS	100

Table 1

Six different glucose concentrations were then made as depicted in table 2. PBS was used as a solvent.

Final glucose concentration, (μg/ml)
0.50
1.00
.25
1.50
1.75
2.00

Table 2

Protocol
1. 50 μl of glucose solution was added into each well. Samples were run in triplicates.
2. Tube containing master mix was placed into spectrophotometer.
3. 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 μl of master mix was added into each well.
Results
1. 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
1. Reaction rate did level off after measuring absorbance for 5 min. However, colour intensity was still very poor.

Inducible Gene Switch

Important dates:
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 (i.e. absorbance measured every 2 sec for 5 min). However this time we doubled the concentration of each reagent that made up the master mix (table 3).

	Final reagent concentration, (μg/ml)
GOx	120
HRP	20
ABTS	200

Table 3. Master mix

Result

Increasing the concentration of master mix reagent did not yield intense colour changes (figure 1)

Figure 1: Colour change observed following absorbance of oxidised ABTS. Master mix containing: GOx, HRP and ABTS was added to 6 different concentrations of glucose.

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).

	Final reagent concentration, (μg/ml)
GOx	625
HRP	62.5
ABTS	625

Table 4. Master mix

Result

Strongly visible green colour was not observed following today’s experiment

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 (i.e. absorbance measured every 2 sec for 5 min).

Final glucose concentration, (μg/ml)
0.50
1.00
.25
1.50
1.75
2.00

Table 5

Result

At the end of our experiment we noticed very intense colour change as shown in figure 2.

Figure 2: Colour intensity following addition of master mix (ABTS, HRP and H2O2)

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) (table 6).

Final ABTS concentration, (μg/ml)
1
5
10
15
20

Table 6

Measured absorbance of water at 420 nm
Measured absorbance of each of the reagents at 420 nm

	Concentration (μg/ml)
HRP	0.25
HRP	2.50
H₂O₂	5.08

Table 7

Set up the reaction with all 4 reagents. Measured absorbance every 25 sec for 900 sec. Run samples in triplicates

	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

Table 8. Volume of reagents to add per well

	Stocks
H₂O₂	0.05g/ml
ABTS	0.0025g/ml
HRP	0.00025g/ml

Table 9. Concentration of stocks used

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.