Cloning

Manipulating DNA and inserting it into different chassis organism is a crucial step to begin synthetic biology, here you can find the different part that we cloned for each step of our experiments.

1. Mimicking Vibrio sp. presence with an engineered E .coli

Part Part:BBa_K2278001 and Part: Part:BBa_K2278002 were constructed to produce respectively C8-CAI-1 and CAI-1 in E .coli. The cqsA from V. harveyi (i.e. Vh-CqsA) or V. cholerae (i.e. Vc-CqsA) coding gene was placed under the control of the pLac promoter (part: Part: BBa_R0040), a strong RBS ( Part: BBa_B0034), and a terminator (( Part: BBa_B1006) (Figure 1). IDT performed the DNA synthesis and delivered the part as gBlock. The constructs were cloned by conventional ligation into the pSB1C3 plasmid and then transformed into E .coli DH5α or TopTen strain. Three transformants of each were tested (Figure 2). Sequencing (figure 3) revealed that the VhCqsA construction slightly differs from the initial design, with a loss of the 9 last amino acids of the protein (position 382 to 391; confirmed on two different runs).

2. E .coli producing C8-CAI molecules can be sensed by V. harveyi

No cloning were made there.

3. Modification of V. harveyi to detect both C8-CAI-1 and CAI-1

This part (Figure 4) was not submitted in the registry and was created to implement the protocol of triparental conjugation in V. harveyi. The part: Part:BBa_J04450 containing the LacI promoter + the RFP encoding gene + a terminator was cloned by conventional ligation into pBBR1MCS-4 and pBBR1MCS-5 (two conjugative plasmids) and then transformed into E .coli Top10 (Figure 5). These plasmids were then used conjugated into V. harveyi by triparental conjugation to validate both the biobrick in V. harveyi and our conjugation protocol.

This part (Figure 6) was not submitted on the registry and was created to allow V. harveyi to recognize both the C8-CAI-1 and CAI-1 from V. cholerae . This part includes the complete cqsS* gene driven by a constitutive promoter ( Part:Bba_J23106 ) and the tetracyclin repressor under the control of pQRR4 promoter ( Part:Bba_K1311017 ) which activation depends on CqsS* detection of C8-CAI-1 and CAI-1. Strong RBS ( Part:Bba_BBa_B0034 ) and terminator ( Part:Bba_BBa_B1006 ) were surrounded the ORF. IDT performed the DNA synthesis. Because of its length, it delivered the part as two gBlocks subparts to assembly. Both subparts were cloned separately by conventional ligation into pBR322, then transformed into E .coli Stellar strain. The final part was cloned by another conventional ligation of the first subpart into the second one, and transformed into E .coli Top10. Six transformants were tested (Figure 7).

4. Production of diacetyl to establish communication between prokaryotic and eukaryotic cells

( Part:BBa_K2278011 (Figure 8) was constructed to test the diacetyl production in E .coli before implementing the pathway in V. harveyi. The gene als encoding for the acetolactate synthase responsible for the diacetyl production, was placed under the control of the pTet promoter ( Part: BBa_R0040), a strong RBS ( Part: BBa_B0034), and a terminator ( Part: BBa_B1006). IDT performed the DNA synthesis and delivered the part as gBlock. The construct was cloned by conventional ligation into the pSB1C3 plasmid and transformed into E .coli Top10 strain. 5 transformants were tested (Figure 9).

5. P. pastoris is able to detect diacetyl from the environment

This part (Figure 10), not submitted to the registry, was created to allow P. pastoris to sense diacetyl and produce AMPs in response. This part includes Odr-10 receptor driven by a constitutive yeast promoter pGAP ( Part:BBA_K431009) and flanked by a kozac sequence ( Part:BBA-J63003) and a stop sequence ( Part:BBA-J63002). It also includes cOT2-coding-gene (AMP) under the control of pFUS1 promotor ( Part:BBA_K1072023) inductible by the Ste12 protein activated by Odr-10 pathway when diacetyl is detected. The gene of cOT2 is flanked by kozac sequence ( Part:BBA-J63003) and a stop sequence ( Part:BBA-J63002). This part has been successfully integrated in P. pastoris’genome (Figure 11).

A second part was made by replacing the cOT2 gene by the RFP to check if Odr-10 was functional in vivo (Figure 12) and this part has also been successfully integrated (Figure 13).

6. P. pastoris is able to produce functional antimicrobial peptides

Parts ( Part:Bba_K2278021 (D-NY15 gene), ( Part:Bba_K2278022 (Leucrocine I gene) and ( Part:Bba_K2278023 (cOT2 gene) were built to test the production of AMPs in P. pastoris (Figure 14). Genes encoding for Leucrocine I, D-YN15, and cOT2 were placed under the control of an alpha factor signal ( Part:BBA_K1800001). IDT performed the DNA synthesis and delivered the part as gBlock. The constructions were cloned by conventional ligation into the pPICZα yeast vector containing pAOX1 or pGAP) (Figure 15) and integrated into the yeast genome (Figure 16). Sequencing (Figure 17) revealed that the AMP constructions do not contain any mutation.

To prove the functionality of the pGAP promotor, the RFP gene has been cloned in the pPICZα-D-NY15 instead of the D-NY15 gene (Figure 18) and this part has been integrated into the genome. This work has been performed by the IGEM team of Vienna as collaboration and they send us the engineered strain as well as the proof of integration by PCR colony gel (Figure 19).