Difference between revisions of "Team:NAWI-Graz/Proof"

Vector Assembly

Our vector had to be assembled from the scratch. We started with gBlock from IDT. Those gBlocks already had the BioBrick pre- and suffix included. As vector backbone the pUC19 plasmid was used. Before doing the assembly we had to make sure we have the correct sequence. We carried out a blunt-end ligation of every gBlock to a commercial pJET 2.1 vector for sequencing. After sequencing we picked the appropriate clones to do PCR reactions in order to get the correct fragment from the pJET vector. After cleaning the PCR products we digested the pUC19 with EcoRI and PstI. The first insert was cut with EcoRI and SpeI, the second insert with XbaI and PstI. Afterwards the ligation of Insert1 (toxin_dammet), Insert2 (colour_antitoxin) and Vector was done.

Characterisation

After the assembly we transformed our plasmid to E.coli XL1 and plated them on LB agar plates. The first few attempts showed really bad growth, so we started to add glucose to the LB plates at a concentration of 25mmol/l in order to have the tac promoter repressed.

The colonies we got were used for an Over Night Culture (ONC). To see if E.coli has the correct plasmid we performed a plasmid preparation and used EcoRI for restriction analysis. The next step was sequencing to see if the plasmid was all correct or if there happened any mutations during the growth phase that led to a non-functional toxin. After we got our sequencing results confirmed we scaled up the ONC to 500ml and started induction at OD600 0.6 to see if the protein was expressed or not. We used SDS-PAGE to compare induced against non-induced protein and to figure out if our proteins are soluble or non-soluble, we compared the supernatant against the cell lysis. Because SDS-Page gives no real proof if it was exactly our protein we did mass spectrometry.

Microbial Part

For the final task we had to force our E.colis to combate each other, in different ways. The first method is using LB plates with 0.1mmol IPTG for induction. We pre mixed colonies from red and blue in a reaction tube and plated them. The second method was done by using an inoculation loop and striking the different strains above each other. This time we let them grow first and used 100µl of 0.1mmol IPTG to induce the cells afterwards. A third idea was to let both strands grow in liquid LB medium overnight and plate them the next day. After one more day the number of surviving red and blue colonies can be counted.

General Protocols

Antibiotic stock solution
Ampicillin (10mg/ml) in H2O sterile filtrated
Chloramphenicol (34mg/ml) in ethanol

LB medium
Plates:
32g /l Invitrogen Lennox l Agar
Autoclave at 121°C for 20min

Liquid:
10 g/L Trypton
5 g/L Yeastextract
5 g/L NaCl
Autoclave at 121°C for 20 min

Restriction Endonuclease Reaction (double digestion)
1μL restriction endonuclease 1 FastDigest
1μL restriction endonuclease 2 FastDigest
2μL FD- Buffer
800-1000ng template DNA
Add H2O to reach a total volume of 20 μL

Ligation:
1µl T4 Buffer
0,5µl T4 Ligase
100µg Backbone
Insert to a ratio of 1:1
Add H20 to a total volume of 10µl

PCR reaction

Components 20 μL reaction volume 50 μL reaction volume 5x Phusion HF buffer 4 μL 10 μL 10 mM dNTPs 2 μL 5 μL Forward Primer (10 μM) 1 μL 2.5 μL Reverse Primer (10 μM) 1 μL 2.5 μL DMSO* 0.6 μL 1.5 μL Phusion DNA polymerase 0.2 μL 0.5 μL DNA 40-200 ng 40-200 ng H2O add to a volume of 20 μL add to a volume of 50 μL *optional

MEGAWHOP Cloning
10µl of 5x Buffer
5µl dNTPS
150ng of mega primer
50ng tamplate DNA
0.5µl Phusion Polymerase
Add H2O to 50µl

PCR profile
98°C for 5min

98°C for 30sec
58°C for 30sec 25cycles
72°C for 3min

72°C for 10min
4°C

To get rid of the parental DNA use 1,5µl DpnI and incubate at 37°C for 2 hours.