Team:WHU-China/Protocol/Lab1
Protocol of molecular biology laboratory
I． Protocols for Light control system
Material:
(1) DNA element :
1. Ccar and Ccas : They could form heterodimer as transcription factor to activate the Cpcg (light control promotor) under green light. And the heterodimer is unstable under blue light.
2. Lac operator : They could block the expression of downstream genes and regain the promotor ability after inducted by IPTG
3. Cpcg : The promotor could be activated by heterodimer made up of Ccar and Ccas
4. TetR : TetR could bind to the tetracydine operator tetO to inhibit transcription .
5. TetO : TetO could be inhibited by tetR
(2) Plasmids :
1. Prefix + lac operator + Ccar + Ccas + suffix
2. Prefix + lac operator + Ccar + Ccas + Cpcg (light control promotor) + green fluorescence protein + suffix
3. Prefix + lac operator + Ccar + Ccas + Cpcg (light control promotor) + TetR + TetO + green fluorescence protein + suffix
4. Prefix + constitutive promotor + GFP + suffix (The positive control in Interlab)
Experiments:
1. Transform the plasmids we have constructed into E.coli BL21 and select the single colony to cultivate in 10ml LB medium overnight.
2. Transfer 1ml medium after cultivating overnight into the 9ml sterilization LB medium
3. Cultivate the medium for 2 hours in 37℃
4. Add the IPTG to activate the gene expression
5. Get samples of medium after cultivating in 0h, 2.5h and 5h . Totally 3 samples each plasmid. Measure the fluorescence intensity of these samples ( activated with 480 nm light and measure in 530nm light)
6. After inducing for 5 hours, cultivating the transformed E.coli under green light, red light and darkness for 2 hours.
7. Get samples after activated by different lights and then transform the green light into red light, vice versa , but bacteria in darkness keep dim.
8. Get samples after transforming the light for 1 hour.
II． Protocol of measuring the total phosphorus.
Kit: made by HANGZHOU LOHAND BIOLOGICAL Co. Ltd.
Principle: Ammonium molybdate spectrophotometric method
Steps:
1. Add one pack of Agent1 to 5ml desired liquid*.
2. Shake it thoroughly to mix the solution.
3. Put it into a autoclave setting at 121°C for 20 minutes to decompose the matter in liquid.
4. When cooling down, add a pack of Agent2 and mix it thoroughly again.
5. Add 7 drops of Activing AgentP .
6. Vortex the solution for 1 minute.
7. Use a spectrophotometer to measure OD value at 700nm.
*The liquid should be dilute to make the total phosphorus at 1-20mg/L.
III．Protocols of PolyP staining:
1. Make a bacteria smear. Fire fixed.
2. Use solution A(80ml) dye it for 5 minutes
3. Wash under distilled water
4. Use solution B(80ml) dye it for 1 minute.
5. Wash under distilled water
6. After dry, use microscopy exam. The body of bacteria turn green and the metachromatic granules turn black blue.
IV． Protocols of verification of bio-safety—kill switch
Material:
(1) DNA element :
1. parD/parE: parD is a toxin-encoding gene that encodes a toxin that inhibits DNA helicase activity, thereby inhibiting bacterial growth. parE is an anti-toxin-encoding gene that encodes an anti-toxin protein that can bind the parD-encoded toxin to make it lose toxicity, but the anti-toxin protein is unstable and easily degraded by serine proteases.
2. Lac operator : They could block the expression of downstream genes and regain the promotor ability after inducted by IPTG
3. MazF/MazE: MazF is a toxin-encoding gene that encodes toxin proteins that express degradation of mRNA to inhibit translation, thereby killing bacteria. MazE is an anti-toxin-encoding gene that encodes an anti-toxin protein that can bind to the MazF-encoded toxin to make it lose toxicity, but the anti-toxin protein is unstable and easily degraded by serine proteases.
4. Theophylline ribose switch: When theophylline is present, the theophylline and the riboswitch are combined, so that the nearby RBS cannot be further used, so that the translation cannot be completed.
5. TetO : TetO could be inhibited by tetR
(2) Plasmids:
1. Prefix + lac operator + parD+ riboswitch +parE + suffix
2. Prefix + lac operator + MazF + riboswitch + MazE + suffix
Experiments:
1. Transform the plasmids we have constructed into E.coli BL21 and select the single colony to cultivate in 10ml LB medium overnight.
2. Transfer 1ml medium after cultivating overnight into the 9ml sterilization LB medium
3. Cultivate the medium for 2 hours in 37°C
4. Take 30ul of the inoculum to the theophylline concentration of 0, 0.1, 0.2, 0.4, 0.8, 1.6 (units g / L) and 0, 0.5, 1, 2, 4, 6, 8 (units g / L) 10 ml of LB liquid medium
5. Cultivate at 37 degrees, shaker 200 rpm culture
6. The OD600 was measured for 6h and 12h, respectively, to determine the number of viable bacteria.