Expressions Test:

We conducted an expressions test with a Barstar model protein. Barstar is a small protein, sythensized from Bacillus amyloliquefaciens. Miche already prepared this protein, when he inserted a stop codon in its sequence. Due to the insertion of a synthetic amino acid P-Benzyl –L-Phenylalanine (Bpa), the stop codon could be over-looked.

Expectation:

if the expression functions correctly – that would mean that the stop codon will be over-looked, and Bpa will be inserted in its place – a band must appear at approximately 10kDa.

Implementation:

• Picking of individual colonies and application of a liquid culture

• control of growth by measuring OD at 600 nm

• when the OD value is at approximately 0.6, Induction of protein expression with IPTG

• Harvesting the cells after 4.5 h Expression at 30° C

Result:

there is no visible band in the gel at 10kDa. This means that the stop codon was not skipped and the synthetic amino acid was not inserted.

Filtration of GFPuv:

Using the His-Tag method, a DNA sequence of the protein to be isolated is amended with the codon of a His-Tag (His-Linker, made of six histidine residues). This His-Tag is bound specifically to column materials with bivalent nickel ions. This specificity allows for only fusion proteins to bond with the column material.

Expectation:

With the help of the His-Tag method, it should be possible to effectively filter the GFPuv.

Implementation:

• Opening the cell membrane with ultrasound

• centrifuging the cell components away

• preparing the nickel column

• Filtration of the protein out of the Lysat (binding and elution of the protein)

• Measuring the protein concentration

• preparing the probe for the mass spectrometer

• measuring the fluorescence and UV spectrum

Picture 1

Picture 2

Result:

The first photo of the SDS-gel was made under UV light. Here the bands are almost impossible to detect. For the second photo the proteins were already colored. S: standard protein marker (5µl), P: cell pallet; D: discharge; 1-3: Wash cycles 1-3; GFP: filtered protein/GFP. In the second photo, one can clearly see how many proteins were disposed of in the first discharge and wash cycle. The column was already saturated by the second and third wash cycles, which is why there are such low amounts of GFP in the wash solution. This is visible via the two bands with 25 kDA that are at the same level as the filtered GFP. The thick band in the GPF row shows that the filtration worked

Filtration of Plasmid- DNA/ Restricted Metabolism:

Expectation:

Cleanly filtered Plasmid- DNA and bands of the correct size in the DNA-gel

Implementation:

• dissolve the cell pellet

• cells are chemically opened up, through the addition of another diluent buffer carbohydrates, proteins etc. are removed

• with a centrifuge the separated DNA can be directly transferred to the prepared columns without the separated material

• The DNA is washed with isopropanol and ethanol after being equilibrated

• the dried DNA is dissolved in water

• the concentration can be determined with Nanotrop

• restricted metabolism

• placed onto DNA-gel

Result:

there is a defined band visible in every probe. Probes 3,4 and 5 had the restriction enzyme cut two times, which can be determined by the two bands in each line (at 4,500 and 800pb).

Transformation of a Shuttle Vector in Bacillus subtilis:

Expectations:

The Bacillus subtilis strain, in which the gene for amylase production is "turned off", should take on a shuttle vector with an amylase gene and be thusly prompted to produce amylase once again

Implementation:

• The Bacillus subtilis culture is prepared

• before the cells reach the stationary phase, they are harvested (after 5 h)

• EDTA will be added to a part of the cells (helps with the later acceptance of vectors)

• Plasmids are added (after two hours at 37 °C in the shaker, the cells should have accepted the plasmids and thereby become resistant to antibiotics)

• cells are placed on a plate – the colonies are allowed to build over night

Ergebnis:

The cell growth is presented in the following diagram:

no colonies are building up on the plates, which means that the vector was not accepted and the cells were not resistant to antibiotics. One reason could be that we used EDTA and not EGTA, as was stated in the protocol.