Team:IISER-Pune-India/Parts

Parts Overview

We have designed 32 parts in total, 4 of which were designed by modifying existing parts in the registry. These include human and parasite protein domains for some of the interactions important for Malaria caused by Plasmodium falciparum, potential inhibitors to these interactions and composite parts that act as drugs that could potentially inhibit the above-mentioned interactions.

We have designed basic biobricks that produce the protein domains of ICAM-1 (BBa_K3582000), CD36 (BBa_K3582002), Basigin (BBa_K3582301) and EPCR(BBa_K3582101) which are human proteins thought to be important for the spread of the infection. Furthermore, we designed biobricks that produce the protein domains of PfRH5(BBa_K3582302), CIDRa domain from HB3var03 PfEMP1(BBa_K3582202), CIDRa domain from IT4var07 PfEMP1 (BBa_K3582102), the CIDRa domain from MCvar1 PfEMP1(BBa_K3582001) and the DBLb domain of PF11_0521 PfEMP(BBa_K3582004), which are parasite proteins that interact with the above human proteins.

We have also designed 2 inhibitory sequences (BBa_K3582024 and BBa_K3582027) for inhibiting the ICAM1-PfEMP1 interaction, 3 inhibitory sequences (BBa_K3582012, BBa_K3582013 and BBa_K3582014) for inhibiting the CD36-PfEMP CIDRa Domain Interaction, 2 inhibitory sequences (BBa_K3582203 and BBa_K3582206) for inhibiting the PfEMP1-EPCR interaction and 1 inhibitory sequence (BBa_K3582303) for inhibiting the PfRH5-Basigin Interaction.

We wanted to use the N intein (BBa_K1362400), C intein (BBa_K1362401) and Bsa1 recognition site(BBa_K1362424) designed by the Team Heidelberg 2014 to circularise our cyclotide. However, we ran into some problems while trying to do so. These biobricks were compatible with Golden Gate Assembly but were not compatible with RF-cloning/Gibson’s Assembling technique. Moreover, the nucleotide sequence of the inteins was not in a set of 3 which led to the ORF being read wrong and problems associated with translation arose as a result. Our team has made some improvements to these new parts and created the modified N intein (BBa_K3582022), C intein (BBa_K3582020) and the Bsa1 recognition site (BBa_K3582021). We made modifications by removing the unnecessary residues from the old biobricks and making our new biobricks compatible with Gibson’s Assembly.

One of our most notable modifications is the modified pet28+ vector (we modified the pet28+ vector of the iGEM in which we removed the unnecessary parts such as the 6x His-tag and the Thrombin site as these parts could interfere with protein expression and purification. This modified biobrick also satisfies the RFC10 criteria while conserving all the other important features of the vector.[1][2] This part can be used by other teams as it is suitable for RF-cloning/Gibson’s Assembling technique and many problems associated with protein synthesis can be avoided.

We have designed composite parts that consist of the modified N intein, C intein, and BSA1 recognition site, Bsa 1 Reversed recognition site (BBa_K2333015), Strep-tag and the inhibitory sequence for the specific composite part. These composite parts produce the cyclotide with an inhibitory sequence in the 6th loop. Each composite part acts as a ready-made drug that potentially inhibits the interactions important for the spread of the infection. The illustration for one of the composite biobricks (BBa_K3582005) is shown below:

We have designed a biobrick for the cyclotide Kalata B1 with a Strep II tag (BBa_K3582023). This construct is designed in a way as to allow the insertion of peptide sequences into the 6th loop of the Kalata B1 construct. The cyclotide backbone is known to be extremely stable and resistant to degradation[3]. Other teams can use this construct in the future as a stable protein scaffold for the insertion of a peptide of interest. This becomes becomes especially useful while designing and experimenting on different peptide sequences.

We have also added new information to the experience page of the following existing parts:

  1. BBa_K1362401 designed by the Team Heidelberg 2014
  2. BBa_K1362400 designed by the Team Heidelberg 2014
  3. BBa_K1362424 designed by the Team Heidelberg 2014
  4. BBa_K2141000 designed by the Team Istanbul_Tech 2016

Parts Table


<groupparts>iGEM20 IISER-Pune-India</groupparts>


References

1: plasmid.med.harvard.edu/PLASMID/GetVectorDetail.do?vectorid=319

2: dnasu.org/DNASU/GetVectorDetail.do?vectorid=319

3: Sancheti, H., & Camarero, J. A. (2009). “Splicing up” drug discovery. Cell-based expression and screening of genetically-encoded libraries of backbone-cyclized polypeptides. Advanced Drug Delivery Reviews, 61(11), 908–917.
DOI: 10.1016/j.addr.2009.07.003





Team IISER Pune India