Team:Tec-Chihuahua/Parts

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Basic Parts Used

Promoter T7 and RBS [BBa_K525998]

This unregulated T7 promoter, which has an integrated RBS, has high levels of transcription when the T7 RNA polymerase is present; thus, in order to induce expression of this BioBrick™ under the control IPTG will be used.

PelB leader sequence [BBa_J32015]

The pelB leader sequence is a sequence of amino acids which, when attached to a protein, directs the protein to the bacterial periplasm. Protein secretion can increase the stability of cloned gene products. It was shown that the half-life of the recombinant proinsulin is increased 10-fold when the protein is secreted to the periplasmic space.

Defensin 1 [BBa_K1104301]

Defensin 1 is synthesized in salivary glands and it plays an important role in the social immunity system of the bee, meaning that it is commonly passed through generations. Defensins unpolarize inner membranes by causing an ionic outer flow using preexistent channels. Apart from these processes, they are responsible for respiratory inhibition 3 minutes after they are added by directly attacking the respiration chain components.

Abaecin [BBa_K1104300]

Antimicrobial peptide that shows activity against both Gram-negative and positive bacteria. When being alone, it shows no activity against E. coli, at least at concentrations up to 200 μM. Hymenoptaecin a native peptide showed activity starting at 2μM concentration. When working together, helps by opening channels for the abaecin, which then gets to attack the DnaK, getting to interfere with a proper folding of the proteins.

T1 from E. coli rrnB [BBa_B0010]

A transcriptional terminator consisting of a 64 bp stem-loop. For the construction of our genetic circuitry, an efficient and reliable terminator was needed.

New Additions to the Parts Registry

Modified 6xHis-tag [BBa_K2834002]

A silently mutated amino acid motif in proteins that consists of six histidine (His) residues, often at the N- or C-terminus of the protein. Polyhistidine-tags are often used for affinity purification of polyhistidine-tagged recombinant proteins expressed in Escherichia coli and other prokaryotic expression systems.

Apidaecin [BBa_K2834000]

It affects mainly Gram-negative bacteria, killing it impressively fast. Its mechanism of action is pretty unique. It has a total lack of pore-forming activity and its efficiency depends on the enantiomer used, meaning it has stereospecificity. Apidaecin gets to the ribosome 70s and the DnaK protein, leading to a protein synthesis inhibition when getting in contact with the ribosome, however, DnaK is the final target. This DnaK has an essential role in the folding of the proteins and in the DNA replication process, especially in the lambda phage.

Composite Parts in our Creation

(T7 promoter + RBS) + (PelB) + (Defensin1) + (6x His-Tag) + (T1 terminator)

[BBa_K2834005]
(BBa_K525998) + (BBa_J32015) + (BBa_K1104301) + (BBa_K2834002) + (BBa_B0010)

This BioBrick™ contains the necessary genetic circuitry to be induced by IPTG and express the Defensin 1 peptide which will be transferred to the periplasmic region by the pelB. Post sonication this peptide can be isolated with the 6x His-Tag using an antiHis column.

(T7 promoter + RBS) + (PelB) + (Abaecin) + (6x His-Tag) + (T1 terminator)

[BBa_K2834006]
(BBa_K525998) + (BBa_J32015) + (BBa_K1104300) + (BBa_K2834002) + (BBa_B0010)

This BioBrick™ contains the necessary genetic circuitry to be induced by IPTG and express the Abaecin peptide which will be transferred to the periplasmic region by the pelB. Post sonication this peptide can be isolated with the 6x His-Tag using a antiHis column.

(T7 promoter + RBS) + (PelB) + (Apidaecin) + (6x His-Tag) + (T1 terminator)

[BBa_K2834003]

(BBa_K525998) + (BBa_J32015) + (BBa_K2834000) + (BBa_K2834002) + (BBa_B0010)

This BioBrick™ contains the necessary genetic circuitry to be induced by IPTG and express the Apidaecin peptide which will be transferred to the periplasmic region by the pelB. Post sonication this peptide can be isolated with the 6x His-Tag using a antiHis column.

References

1. Ilyasov, R. A., Gaifullina, L. R., Saltykova, E. S., Poskryakov, A. V., & Nikolaenko, A. G. (2013). Defensins in the honeybee antiinfectious protection. Journal of Evolutionary Biochemistry and Physiology, 49(1), 1–9. https://doi.org/10.1134/s0022093013010015
2. Cociancich, S., Ghazi, A., Hetru, C., Hoffman, J. A., & Letellier, L. (1993). Insect Defensin, an Inducible Antibacterial Peptide, Forms Voltage-dependent Channels in Micrococcus luteus. The Journal of Biological Chemistry, 268(26), 19239-19245
3. Shen, X., Ye, G., Cheng, X., Yu, C., Hu, C., Allosaar, I. (2010). Characterization of an abaecin-like antimicrobial peptide identified from a Pteromalus puparum cDNA clone. Retrieved from: https://www.sciencedirect.com/science/article/pii/S0022201110001114
4. Castle, M., Nazarian, A., Yi, S., Tempst, P. (1999). Lethal effects of apidaecin on Escherichia coli involve sequential molecular interactions with diverse targets. Retrieved from: http://www.jbc.org/content/274/46/32555.full.pdf
5. Cociancich, S., Ghazi, A., Hetru, C., Hoffman, J. A., & Letellier, L. (1993). Insect Defensin, an Inducible Antibacterial Peptide, Forms Voltage-dependent Channels in Micrococcus luteus. The Journal of Biological Chemistry, 268(26), 19239-19245