Summary Techniques in molecular bacteriology

Molecular Basis of Bacterial Infections Evelien Floor



Techniques in molecular
bacteriology
We found our immune evasion proteins via genomic predictions. Immune evasion proteins are
usually quite small (< 50 kDa). We filtered for secreted proteins with a secretion signal and a small
ORF. Genomic localization is also important in this prediction because immune evasion proteins can
be close to each other in the genome.




Laboratory steps from DNA to protein:
 PCR/gBlock
 Cloning in expression vector
 Transformation into E. coli
 Colony PCR
 Sequencing
 Transformation in E. coli expression strain
 Transfection in eukaryotic cells

A disadvantage of the use of E. coli is that post translational modifications as glycosylation and
phosphorylation aren’t present. Therefore, the use of eukaryotic cell lines (HEK, CHO) is beneficial.
Post translational modifications provide correct folding and function. In eukaryotic cells signal
sequences for secretion are also present. However, these cells are expensive.
Functional studies:
 Cell interaction: receptor
 Other interaction partners
 Enzyme function
 Structural analysis
o Crystallography
o NMR
 Drug design




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, Molecular Basis of Bacterial Infections Evelien Floor


Laboratory steps
PCR
The template for PCR is genomic DNA of a potential immune evasion protein. The targets are
selected by signal sequences and size, known immune evasion proteins are excluded. For PCR specific
primers for the gene of interest are required. The first 25 and last 25 nucleotides of the genes are
used for PCR.
gBlock
gBlocks are used so you don’t have to do the PCR yourself, this is because all bacteria express amino
acids differently. The gBlock can be ordered and you will receive double stranded DNA of any
sequence you like.
Cloning in expression vector
The signal peptide is cleaved off when a protein is secreted, therefore it isn’t needed in the plasmid.
Only the sequence of the secreted protein is put into the vector. The vector consists of an origin of
replication where replication is initiated. This origin is species specific and can be chosen to express
high or low copy numbers. Furthermore, there is a promotor in the expression vector where
transcription of the gene can be initiated by RNA polymerase. In our case the T7 promoter is used
which can be switched on.
The expression vector also consists of a ribosome binding site and KOZAK sequence for initiation of
protein translation. Then there is a multiple cloning site (MCS) with the gene of interest. This MCS is
followed by a transcription termination site and a selectable marker. The selectable marker is most of
the time an antibiotic resistance gene for selection.




Cloning of a PCR product into a vector is also called
Gibson assembly. The PCR product with flanking
sequences corresponding with the plasmid are added to
the plasmid with a Gibson assembly mix. The Gibson
assembly mix consists of exonuclease, DNA polymerase
and DNA ligase. Exonuclease cleaves off the flanking
sequences and DNA polymerase closes the gaps. In final,
DNA ligase seals the nicks.
A plasmid needs to be closed for replication. To test
whether the plasmid is closed a control with water is
performed. The plasmid will then still be open, and the
bacteria shouldn’t be able to grow. If they do grow,
something went wrong.

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