Project Results

Design of DAMP4 expression cassette - We made our design as illustrated in the following figure:

The part was ordered as a gBlock fragment and cloned into pSB1C3 (part BBa_K3208000)

• Design of arabinose-inducible expression system - we assembled parts BBa_I0500 and BBa_B0032 into one module. To prove its functionality, we cloned a red chromoprotein CDS after it and made expression experiments. Everything functioned as expected:
  
• The DAMP4 cassette was cloned under the control of the arabinose-inducible expression module. Next, we performed a series of expression experiments using the empty vector (with a staffer peptide) and vectors with known AMPs (pexiganan and SAAP-148). Results are illustrated in the following figure:
  Legend: The thin band shows that the arabinose promotor is too weak. This is the reason why we switched it with the T7 promotor for our AMP expression blocks.
• Creating a list of known AMPs active against Gram-positive/ Gram-negative bacteria. (2 FASTA files with all AMPs from DBAASP (10.09.2019) that fulfill the following criteria: 10-30 amino acids in length, monomeric molecules, no unnatural amino acids, no C/N-terminus modifications, active against Gram-positive/Gram-negative bacteria).
  AMP Anti Gram -
  AMP Anti Gram +
• Identification of putative AMPs from the genome of the great white shark. We successfully identified 9 putative AMPs.
  
  We extracted their sequences and optimized them for expression in E.coli, using the IDT tool. Their AA sequences and the corresponding DNA blocks are given in the following file
  Carharins
• Design of expression cassettes for all putative carharins. All expression cassettes use an identical design that is illustrated in the following figure.
  
  Тhe separate constructs were submitted as BioBrick parts with numbers BBa_K3208001 to BBa_K3208009.
• Cloning of the putative carharins: - gBlock fragment amplification:
  Legend: 1 - 1kb+ Ladder; 2 - carharin 1; 3 - carharin 3; 4 - carharin 4; 5 - carharin 5; 6 - carharin 6; 7 - carharin 7; 8 - carharin 8; 9 - carharin 9; 10 - carharin 10
  
  - Cloning them into pSB1C3 (all without 1 & 8)
  
  
  - Colony PCR confirmation of correct clones
  Legend: 1 - 1kb+ Ladder; 2 - carharin 2; 3 - carharin 3; 4 - carharin 4; 5 - carharin 5; 6 - carharin 6; 7 - carharin 7; 8 - carharin 9; 9 - empty well; 10 - negative control
  
  - Measurement of the growth kinetics after induction of the AMP expression
  Legend: After induction, all putative AMPs show an inhibitory effect over the cell growth to a given extent compared to control. This is a strong indicator that our putative AMPs might have antimicrobial properties against E.coli.