Transformation of plasmids pET 43.1a and pSB1C3 in E. coli DH5-α competent cells, in order to constitute a stock of empty vectors for our further experiments.

Bacteria transformed with pET43.1a did not grow. Bacteria transformed with pSB1C3 did grow. One colony was placed in liquid culture and we let grow overnight.

Midiprep of bacteria containing pSB1C3.

Transformation of our first sequences from Eurofins in E. coli DH5-α:
- 3a_NGF construction Part1 (Seq#1)
- 3a_NGF construction Part2 (Seq#2)
- T7 RIP construction (Seq#8)

All the bacteria were successfully transformed with the different plasmids. One colony from each plate was placed in liquid culture and let grow overnight.

Midiprep of:
- Seq#1
- Seq#2
- Seq#8

Culture of strain HB2151 transformed with pVDL 9.3 (received on 23.07.2018 from Victor de Lorenzo).

Biofilm culture on filter (0.2μm).

Transformations: To create a stock
-seq#5, seq#7 (detection) from Eurofins
-plasmids pET 43.1 and pBR322 (expression plasmids)

Biofilm culture on filter after 24 hours incubation did grow.
Bacteria transformed with pET43.1a did not grow.
Overnight liquid culture of seq#5, seq#7 and plasmid pBR322.

Midiprep of plasmid pVDL 9.3 and measure DNA concentration with nanodrop.

Midiprep of seq#5, seq#7 and plasmid pBR322 contained in DH5α strain and measure DNA concentration with nanodrop.

Preparing cultures for stocks: we set some pre-cultures of seq#5, seq#7 and pBR322, in order to make some stocks to store at -80°C.

Digestion, electrophoresis on agar gel, DNA gel extraction of seq#8 and pBR322.

Ligation of linearized pBR322 and our insert seq#8 with the in-fusion kit and transformation of pBR322 + seq#8 in Stellar competent cells.

Transformation of seq#6 (detection) received from Eurofins in E. coli DH5-α.

Overnight liquid culture of DH5-α with seq#6.

Overnight liquid culture of Stellar competent cells transformed with pBR322 + Seq8.

Transformation of seq#3 (NGF-GFP) from Eurofins and commercial pET43.1a.

Miniprep, digestion and electrophoresis on agar gel of pBR322 + seq#8 from Stellar competent cells and Nanodrop.

Midiprep of seq#6 plasmid from DH5-α and nanodrop.

Digestion, electrophoresis on agar gel and gel extraction of pET 43.1a, seq#1 and Seq#2.

Ligation of linearized pET43.1a and our inserts Seq#1,2 with the in-fusion kit. Transformation in Stellar Competent cells.

Electrophoresis agar gel of the miniprep of the ligated plasmid (pBR322 + seq#8) from Stellar, WRONG ENZYMES USED FOR THE DIGESTION.

2^nd Electrophoresis agar gel of the miniprep of the ligated plasmid (pBR322 + seq#8) from Stellar, using the correct enzymes.

Overnight liquid culture of pET43 1.a.

Overnight liquid culture of Stellar transformed with pET43 1.a-seq#1-seq#2.

Midiprep of seq#3 plasmid from transformed Stellar competent cells.

Digestion, electrophoresis on agar gel and gel extraction of pBR322, seq#5, seq#6 and seq#7.

Ligation of linearized pBR322 and our insert seq#8.

Miniprep of plasmid pET43.1a and nanodrop.

Miniprep of construction pET43.1a + seq#1 + seq#2.

Overnight liquid culture of Stellar competent cells transformed with construction pBR322-seq#5-seq#6-seq#7.

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Miniprep, digestion and electrophoresis on agar gel to verify if seq#1 and seq#2 (NGF) are correctly inserted in our plasmid pET43.1a.

Midiprep of pBR322 + seq#8 (RIP)

Transformation:
- pB3322 + Seq#8 in E. coli BL21 for the expression of our biobrick
- pET43.1a + seq#1 + seq#2 in E. coli BL21 for the expression of our biobrick
- Seq#4 in E. coli DH5α to amplify our plasmid from Eurofins

Bacterial stock of seq# 1,2,3,4,5,6,7,8, and plasmids pET43.1a, pBR322, pVDL9.3 for further use.

Miniprep, digestion, electrophoresis on agar gel and gel extraction for plasmid pBR322, and seq#5, sea#6, seq#7 (detection of S. aureus) in order to obtain high concentrations of purified plasmid and inserts for In-Fusion kit.

Midiprep:
- pBR322 + seq#8
- pET43 + seq#1,2

Results: concentrations of DNA are too low, we will try to obtain higher concentrations with minipreps. Ligation of pBR322 + seq#5,6,7 with the in-fusion kit.

Bacterial stock of S. aureus, E. coli BL21 transformed with pBR322 + seq#8 and E. coli BL21 transformed with pET43.1a + seq#1,2.

Miniprep:
- pBR322 + seq#8
- pET43 + seq#1,2

Midiprep of seq#4

Miniprep, digestion and electrophoresis on agar gel to verify if seq#5,6,7 are correctly inserted in our plasmid pBR322.
Results: CLONING HAS FAILED.

Miniprep, digestion, electrophoresis on agar gel and gel extraction for plasmid pET43.1a, and seq#3, seq#4.
Results: DIGESTION OF SEQ#4 HAS FAILED.

Miniprep of pET43.1a

Miniprep, digestion, electrophoresis on agar gel and gel extraction for plasmid pBR322, and seq#5, sea#6, seq#7 (detection of S. aureus) in order to obtain high concentrations of purified plasmid and inserts for In-Fusion kit.

Antibiotics test on liquid culture in order to evaluate the effect of an overnight culture media containing CARB on a non-resistant strain.

RIP production with different IPTG concentration. Pellet and supernatant of culture are kept separately in order to further test their effect of S.aureus biofilm formation.

Liquid culture of bacteria transformed with pSB1C3 and pBR322 empty plasmid to make plasmid stocks.

Liquid culture of S. aureus

Digestion, electrophoresis on agar gel, gel extraction of pSB1C3 plasmid and all sequences from Eurofins (1,2,5,6,7,8) in order to prepare our sample to send for sequencing and to iGEM judges

Ligation of Seq1+Seq2 and Seq8 in pSB1C3

Miniprep to extract pBR322 and pSB1C3

Digestion and electrophoresis on agar gel of 10 colonies transformed with our cloning product of pBR322+Seq5+Seq6+Seq7 : CLONING HAS FAILED

NGF production induction with different IPTG concentrations.
Pellet lysis and SDS-Page gel of the different samples. After revelation, we have a band around 33 kDa that could be our NGF but it needs to be check.

We plated on a 96-well plate S. aureus with supernatant from E. coli culture producing RIP. Incubation for at least 24 hours.

Digestion, electrophoresis on agar gel, gel extraction and ligation of pSB1C3 plasmid with our 3-insert construction: Seq5+Seq6+Seq7.

Transformation of Seq9 (Kill switch) in DH5 alpha competent cells for amplification.

Results:
Bacteria transformed with our cloning pSB1C3+Seq1+Seq2 and pSB1C3+Seq8 have grown. We put 10 colonies of each in liquid culture to further analyze cloning.

Miniprep, digestion and electrophoresis on agar gel of the colonies from cloning.
Results:
- pSB1C3 + Seq8 :7 out of 10 analyzed colonies get the insert. SUCCESSFUL CLONING
- pSB1C3 + Seq1 + Seq2 : 3 out of the 10 colonies worked. SUCCESSFUL CLONING

Antibiotic test on liquid culture to evaluate the effect of an overnight culture media containing AMP on a non-resistant strain.

Miniprep, digestion and electrophoresis on agar gel of the colonies from pSB1C3+Seq567 cloning.
Results: SUCCESSFUL CLONING

S. aureus was plated on 96-well plate to further test different method to clean the wells

IPTG induction of NGF expression in a 800 mL culture for further analysis.