Revision as of 02:35, 19 November 2018

Autoclave the glass and plastic material (Petri dishes, conical centrifuge tubes, Erlenmeyers) filled ¾ with distilled water to make them free of detergent since any residue is an inhibitor of the growth and transformation of competent cells.
Inoculate 125 mL of LB medium with 1 mL of E. coli cell stock and culture overnight at room temperature. A spectrophotometer or Nanodrop can be used to determine an OD 600nm ≤ 0.3.
Pre-freeze the conical centrifuge tubes.
Transfer the culture to the prechilled conical centrifuge tubes.
Centrifuge for 10 minutes at 3000 g at 4 ° C.
Gently resuspend by vortexing the cell pellet in 40 mL of ice cold CCMB80 buffer.
Hold on ice for 20 minutes.
Centrifuge again for 10 minutes at 3000 g at 4 ° C.
Resuspend by pipetting the cell pellet in 15 mL of ice cold CCMB80 buffer.
Hold on ice for 20 minutes.
Aliquot in cryotubes 2 mL.
Store at -80 ° C.

Preparation of CCMB80 Buffer:

Component	Concentration
CaCl2.2H2O	11.76 g/L
MnCl2.4h2O	3.95 g/L
MgCl2.6H2O	2.03 g/L
KOAc	0.9815 g/L
Glicerol al 10%	100 mL

Note: Adjust the pH to 6.3, sterilize by filtration.

EXPERIMENT 2: Domestication of the gBlocks

In order that the restriction enzymes EcoRI and PstI cleavage at the end of the gBlocks and the cohesive ends be generated, a PCR was run using the first forward 5'-ccggaattcgcggccgcttctag-3 'and reverse 5'-aaaactgcagcggccgctactag-3' to add the nucleotide bases to the N-terminal and C-terminal ends.

PROTOCOL

The PCR Reaction using Q5 High-Fidelity DNA polymerase was setup according the manufacturer´s instructions (New England Biolabs).

Table1. PCR Components

COMPONENT	50 uL REACTION	FINAL CONCENTRATION
5X Q5 Buffer	10 uL	1X
dNTPs (10 mM)	1 uL	200 uM
Primer Forward (10 uM)	2.5 uL	0.5 uM
Primer Reverse (10 uM)	2.5 uL	0.5 uM
Q5 High-Fidelity DNA Polymerase	0.5 uL	0.02 U/uL
Template DNA (50 ng/uL)	0.5 uL	0.5 ng/uL
5X Q5 High GC Enhancer	10 uL	1X
Nuclease-free water	23 uL

Table 2. PCR Conditions

STEP	TEMPERATURE	TIME
Initial denaturation	98°C	30 s
35 ciclos	98°C	10 s
	72°C	25 s
	72°C	25 s
Extensión final	72°C	2 min
Hold	4°C

Experiment 3: Purification of the PCR products of the gBlocks

The objective of purifying the amplicons of the gBlocks was to use it in the process downstream of assembly 3A. The purification was carried out following the manufacturer's instructions (Invitrogen).

Experiment 4 Fusion assembly

Once purified products were obtained from the CBD cipA gBlocks (driven by pLac promoter), BMP2 and sfGFP with basepairs added to the N-terminal and C-terminal ends, we proceeded to perform a fusion assembly to obtain the CBD fusion products cipA-BMP2 and CBD cipA-sfGFP. The methodology involved slight variations to the one described in the Open Wetware site Fusion Assembly.

PROTOCOL

The purified gBlocks CBD cipA, BMP2 and sfGFP were eluted in Elution Buffer E1 (10 mM Tris-HCl, pH 8.5) while the psb1c3 vector was eluted in Nuclease-free water.
Prepare restriction mixes (Table 1) to digest gBlocks

Table 1.

Mix	Component	2 uL reaction
A	10X NEB Buffer 1.1	1.00 uL
	Nuclease-free water	0.47 uL
	EcoRI (15U/uL)	0.13 uL
	AgeI (5U/uL)	0.40 uL
B	10X NEB Buffer 4	1.00 uL
	0.1% BSA	0.32 uL
	PstI (10U/uL)	0.34 uL
	NgoMIV (10U/uL)	0.34 uL
C	10X Buffer O	1.16 uL
	EcoRI (15U/uL)	0.34 uL
	PstI (10U/uL)	0.50 uL

Mix 8.0 uL of purified CBD cipA gBlock (50 ng / uL) with 2 uL of restriction mix A.
Mix 8.0 uL of purified BMP2 gBlock (50 ng / uL) with 2 uL of restriction mix B.
Mix 8.0 uL of purified gBlock sfGFP (50 ng / uL) with 2 uL of restriction mix B.
Mix 3.2 uL of the linearized psb1c3 vector (25 ng / uL) with 0.8 uL of the restriction mix C.
Incubate for 2 h at 37 ° C.
Inactivate the restriction enzymes heating the samples for 20 min at 85 ° C.9. Prepare the ligation mix (Table 2).

COMPONENT	10 uL REACTION
Nuclease-free water	7.0 uL
10X T4 DNA Ligase Buffer	2.0 uL
T4 DNA Ligase	1.0 uL

Mix 4.0 uL CBD cipA digested with 4.0 uL BMP2 digested and 2.0 uL psb1c3 digested vector.
Mix 4.0 uL CBD cipA digested with 4.0 uL sfGFP digested and 2.0 uL psb1c3 digested vector.
Add 10 uL of the ligation mix to each mix (Step 10 and 11).
Incubate overnight at 16 ° C.
Inactivate ligase enzyme heating the samples for 10 min at 65 ° C.
The volume for transformation depends on the total volume of competent cells (See Transformation protocol).

Experiment 5: Transformation

The psb1c3-CBD vectors cipA-BMP2 and psb1c3-CBD cipA-sfGFP obtained by fusion assembly were transformed into E. coli DH5α, BL21 (DE3) cells by heath shock method according to the protocol described in Transformation.

PROTOCOL

Add 70μl of competent cells in a 2ml Eppendorf tube.
Add 8 μl of resuspended DNA in the tube.
Close the 2 ml tubes and incubate on ice for 20 minutes
For thermal shock place the tubes in the water bath at 42 ° C for 45 seconds
Incubate on ice for 2-4 minutes
Add 800μl of SOC medium in each transformation (Table 1)

Table 1. SOC medium composition

Component	Concentration
Triptone	10g/L
Yeast extract	5g/L
NaCl	0,5g/L
MgSO 4	10mM
Glucose	20 mM Glucose * (thermosensitive) Adjust to pH 7 and add these compounds after sterilizing in autoclave

Incubate for 1.3 h at 37 ° C.
Centrifuge the tubes for 3 minutes at 7000 g.
Discard 400μL of the supernatant. Resuspend cells in the remaining 100 μL.
Spread by extension 100 μL of the transformed product in Petri dishes containing LB + chloramphenicol medium (Table 2).

Table 2. Agar LB+cloranfenicol

Component	Concentration
Triptone	10g/L
Yeast extract	5g/L
NaCl	0,5g/L
Agar	15g/L
Chloramphenicol	1 mL of a solution of Chloramphenicol at 25 mg / mL * (thermosensitive) Add this compound after sterilizing in autoclave

Incubate overnight transformations at 37 ° C.

EXPERIMENT 6: Extraction of plasmid DNA

Plasmid DNA was extracted from the bacterial transformation products for subsequent enzymatic digestions and PCR reactions to verify that the psb1c3-CBD inserts cipA-BMP2 and psb1c3-CBD cipA-sfGFP are correct.

PROTOCOL

Add 1.5 mL of bacterial culture in a 2 mL tube.
Centrifuge at 13,000 rpm for 45 seconds at 4 ° C.
Resuspend the pellet in 250 μL cold lysis solution 1 (with RNAase), homogenize vortex.

Table1. Lysis solution preparation

Lysis solution 1	Lysis solution 2	Lysis solution 3
50mM de glucosa 25mM de Tris-HCl pH 8 10mM EDTA pH 8 RNasa 1 ug/mL	0.2 N de NaOH 1% (w/v) de SDS	5M of potassium acetate dissolve with glacial acetic acid and gauge with distilled water

Incubate 2 minutes at room temperature.Add 250 μL of lysis 2, homogenize by inversion.
Incubate 5 minutes on ice.
Add 300 μL cold lysis solution 3, homogenize by inversión
Incubate on ice for 10 minutes.
Centrifuge at 14,000 rpm for 10 minutes at 4 ° C.
Transfer supernatant and add two volumes of absolute cold ethanol or absolute isopropanol, homogenize by inversion.
Incubate at -20 ° C for 20 minutes.l. Centrifuge 14000 rpm for 30 minutes.m. Discard the supernatant.n. Add 1 mL of 70% ethanol and mix by inversion.or.
Centrifuge at 14,000 rpm for 20 minutes.
Discard the supernatant and let it dry for 15 minutes.
Resuspend in 25 μL of water DPC.

Table 2. Materials for Plasmidic extraction

Biological / equipment	Indications
bacterial culture	3 mL of 24 hour liquid bacterial culture
2ml microtubes	Label the tubes with the name of the part or location of the well before beginning
Ice Cube	Fill the bucket with ice and pre-cool the 2mL tubes (5 minutes). Thaw stocks of competent ice cubes (10-15min)
Timer	Preferably with temperature control
2ml tube centrifuge	nuclease-free water

EXPERIMENT 7: Enzymatic digestion of the plasmids containing PSB1C3 as backbone

The psb1c3-CBD plasmid products cipA-BMP2 and psb1c3-CBD cipA-sfGFP were digested with EcoRI and PstI to distinguish the size of the products by means of a 1% agarose gel electrophoresis.

PHASE II: CONSTRUCTON OF THE COMPOSITE PART CBD cipA-ELP

Experiment 1: Amplification of ELP from pET-24a-ELP[V-150]

A PCR reaction was assessed to try amplifying the ELP[V-150] (transition temperature=28.6°C) from the commercial plasmid pET-24a-ELP[V-150] (Addgene). Numerous attempts were done using a set of primers; however, similar electrophoresis patterns with multiple diffuse bands were obtained.

Competent Cells

Protocol of the PCRs tested
The PCR Reaction using Dream Taq DNA polymerase was setup according the manufacturer´s instructions (Thermo Scientific).
Table1. PCR Components

COMPONENT	50 uL REACTION	FINAL CONCENTRATION
DreamTaq PCR Master Mix (2X)	25 uL	1X
Primer Forward (10 uM)	2.5 uL	0.5 uM
Primer Reverse (10 uM)	2.5 uL	0.5 uM
Template DNA (10 ng/uL)	3.0 uL	0.6 ng/uL
Nuclease-free water	17 uL

Table 2. PCR Conditions

STEP	TEMPERATURE	TIME
Initial denaturation	95°C	3 min
Round 1 (5 cycles)	95°C	30 s
	T annealing Round 1*	30 s
	72°C	1.4 min
Round 2 (25 cycles)	95°C	30 s
	T annealing Round 2*	30 s
	72°C	1.4 min
Hold	4°C

Table 3. Primers used in the PCRs tested

PCR	Primers	Primer sequence	T annealing Round 1	T annealing Round 2
1	Forward 1	5'-gaattcgcggccgcttctagatggccggcagttcagggggtgtag-3'	44°C	65.5°C
	Reverse 1	5'-ctgcagcggccgctactagtattaaccggtccagggaactccaac-3'
2	Forward 1	5'-gaattcgcggccgcttctagatggccggcagttcagggggtgtag-3'	45.5°C	67°C
	Reverse 2	5'-ctgcagcggccgctactagtattaaccggtcggccagggaactcc-3'
3	Forward 1	5'-gaattcgcggccgcttctagatggccggcagttcagggggtgtag-3'	44°C	65.5°C
	Reverse 3	5'-ctgcagcggccgctactagtattaaccggtcggccagggaact-3'
4	Forward 2	5'-agaaggaggagtacatatg-3'	42.5°C
	Reverse 4	5'-gatcctgaagattatcacgg-3'

Purification protocol of DNA fragment from agarose gel
Please refer to:
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/purelink_gel_extraction_pcr_combo_man.pdf

Experiment 2: Domestication of the ELP550 amplicon

OOnce primers was purified, the ELP550 obtained with the Forward 1 (5'-gaattcgcggccgcttctagatggccggcagttcagggggtgtag-3 ') and Reverse 2 (5'-ctgcagcggccgctactagtattaaccggtcggccagggaactcc-3') a PCR was run using the ForwardFu primers (5'-ccggaattcgcggccgcttctag-3 ') and ReverseFu (5'-aaaactgcagcggccgctactag-3') to add the nucleotide bases 5'-ccg-3 'and 5'-aaaac-3' to the N-terminus and C-terminus, respectively, and consequently leave space so that the enzymes EcoRI and PstI cut near the ends. The PCR product was purified using the Quick Gel Extraction and PCR Purification Combo Kit according to the manufacturer's instructions (Invitrogen).

PCR SHOW/HIDE PROTOCOL
The PCR Reaction using Dream Taq DNA polymerase was setup according the manufacturer´s instructions (Thermo Scientific).
Table1. PCR Components

COMPONENT	50 uL REACTION	FINAL CONCENTRATION
DreamTaq PCR Master Mix (2X)	25 uL	1X
Primer Forward (10 uM)	2.5 uL	0.5 uM
Primer Reverse (10 uM)	2.5 uL	0.5 uM
Template DNA (60 ng/uL)	1.0 uL	1.2 ng/uL
Nuclease-free water	19 uL

Table 2. PCR Conditions

STEP	TEMPERATURE	TIME
Initial denaturation	95°C	3 min
Round 1 (5 cycles)	95°C	30 s
	54°C	30 s
	72°C	30 s
Round 2 (25 cycles)	95°C	30 s
	57°C	30 s
	72°C	30 s
Hold	4°C

Experiment 4 Fusion assembly

Once purified products were obtained from the CBD cipA gBlocks (driven by pLac promoter), BMP2 and sfGFP with basepairs added to the N-terminal and C-terminal ends, we proceeded to perform a fusion assembly to obtain the CBD fusion products cipA-BMP2 and CBD cipA-sfGFP. The methodology involved slight variations to the one described in the Open Wetware site Fusion Assembly.

Experiment 5: Transformation

See Transformation Protocol, Phase I.

Phase III: Construction of the cellulose production module

Experiment 1: Amplification of LacI from pET-24a-ELP[V-150]

Due to the need of an induce mechanism we decided to use LacI cassette in our assemblies. To obtain this cassette we did a PCR reaction to amplify the LacI (promoter and ORF) from the commercial plasmid pET-24a-ELP[V-150] (Addgene).

Experiment 2: Domestication of the gblocks and the LacI amplicon

In order to obtain specific sequences ends to be digested by the gibson assembly exonuclease, we used specific primers for each sequence to add overhanging ends to the sequence that must be ligated. This overhanging ends were added by PCR with specific conditions for each sequence.

Experiment 3: Purification of the PCR products of the gBlocks

The objective of purifying the amplicons of the gBlocks was to use it in the process downstream of assembly 3A. The purification was carried out following the manufacturer's instructions (Invitrogen).

Experiment 3: Linealization of the psb1C3 and psb1A3 plasmids.

To assemble all the parts to the plasmids with Gibson technique, we made the linearization and removal of the biobrick suffix and prefix from the psb1C3 and psb1A3 through PCR.

Experiment 4: Gibson assembly

Several trials were carried out to assemble two vectors with the cellulose production module as planned. We faced with several problems that occurred during this trial and we could not obtain the assembled vectors that we wanted, but we obtained degraded DNA seen in electrophoresis and this could be due to the high concentration of GC at the ends of the sequences that interfer with the Gibson’s exonuclease cleavage activity.

Phase IV: Characterization of the binding strength of the CBD cipA with an endogenous C-terminal linker

Measurements of remaining fluorescences, after different washes, were done according to the protocol described by the Team Imperial 2014. Briefly, 20g of kombucha cellulose were blended with 125 mL. Then, 100 uL of the mixture were added into each well of a 96-plate reader and dried at 60º for 5.5 hours using a stove. A volume of 100 uL of CBD cipA-sfGFP protein extract was added into each well an incubated at 4ºC overnight. The fluorescence was measured after washing with different substances: dH20, 70% EtOH, Plasma and Stif.

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Componente	Concentración
CaCl2.2H2O	11.76 g/L
MnCl2.4h2O	3.95 g/L
MgCl2.6H2O	2.03 g/L
KOAc	0.9815 g/L
Glicerol al 10%	100 mL

Componente	Concentración
CaCl2.2H2O	11.76 g/L
MnCl2.4h2O	3.95 g/L
MgCl2.6H2O	2.03 g/L
KOAc	0.9815 g/L
Glicerol al 10%	100 mL

Componente	Concentración
CaCl2.2H2O	11.76 g/L
MnCl2.4h2O	3.95 g/L
MgCl2.6H2O	2.03 g/L
KOAc	0.9815 g/L
Glicerol al 10%	100 mL

Componente	Concentración
CaCl2.2H2O	11.76 g/L
MnCl2.4h2O	3.95 g/L
MgCl2.6H2O	2.03 g/L
KOAc	0.9815 g/L
Glicerol al 10%	100 mL

Difference between revisions of "Team:Ecuador/Experiments"

Revision as of 02:35, 19 November 2018

EXPERIMENTS

Index

SHOW/HIDE PROTOCOLO

SHOW/HIDE PROTOCOLO

SHOW/HIDE PROTOCOLO

SHOW/HIDE PROTOCOLO

SHOW/HIDE PROTOCOLO

COMPONENT	3 uL reaction
10X Buffer O	1.74 uL
EcoRI (15U/uL)	0.51 uL
PstI (10U/uL)	0.75 uL