Part Construction

Abstract

For our therapeutic approach we transform our Engineering Design considerations into reality. The generation of an Entry Vector which is capable of generating mRNA sensing and killing switches via in vitro transcription. For intraoperative mRNA therapy, we aim to develop stable and highly efficient sensing and killing switches. By targeting glioma specific alterations, we aim to develop our integrative treatment strategy. With our Part Construction, we presented our results:

☑ assemble of our Entry Vectors pASTERISK for integrating sensor sequences

☑ verification of successfully cloning via Nanopore Sequencing and Sanger Sequencing experiments

☑ optimization of existing protocols by testing different experimental setups to contribute more accessible workflows for future teams

☑ driving our Engineering Cycle to prepare mRNA synthesis via in vitro transcription

We managed to create our pASTERISK Entry Vector for modulable insertion of sensor sequences and payloads to contribute with multifunctional usage. Our Part Pages provides detailed information of our genetically circuit of pASTERISK. Because of the integrated T7 Promoter, the Entry Vector is capable of producing mRNA molecules. By inserting the sensor sequences made with our Software sensor design tool, the sensor cloning site can be opened via HindIII and the sequence can be inserted via Gibson Assembly or Restriction enzyme cloning according to RFC10 standards. In addition, by digest pASTERISK with SphI and SalI, the payload sequence can be changed as well. The modulable design of our plasmids allows different fields of applications and opens more possibilities for future iGEM teams to use adjustable synthetic biology.


ASTERISK-HBA1 / ASTERISK-HBB + T7 promoter and terminator
Figure 1: Circuit diagram of the ASTERISK-HBA1 and ASTERISK-HBB parts in addition with an upstream T7 promoter and downstream T7 terminator. Both of these constructs were cloned into pSB1C3 plasmids.


Preparation of Parts Backbone Plasmids, Expression vectors and cloning fragments

At the beginning of our project, we established chemo- and electrocompetent E. coli for all following transfections and cloning strategies within our project. We verify the transfection efficiency and competenz as well as the quality and selection efficiency. To continue the iGEM tradition of RFC10-based cloning, we decided to utilize pSB1C3 as our main backbone plasmid. The successful transfections were verified via the chloramphenicol resistance and outgrowth in LB media containing appropriate antibiotics resistance. We extracted the plasmid after appropriate outgrowth with different methods including mini spin preparation and midi silica preparation kits.

We successfully transfected and isolated our plasmids for different usage including:

  • pSB1C3 - Backbone plasmid for pASTERISK-HBA1 and pASTERISK-HBB to improve RFC 10 standards
  • pDART VADAR - Plasmid for generating sensor construct that target IRFP720 transcripts in vivo according to Gayet et al. 2023
  • pDART VADAR_05 - Plasmid for constitutive Expression of native IRFP720 transcript
  • pDART VADAR_06 - Entry Vector for cloning of sensor sequencing within target site
  • pcDNA5 - Backbone Vector for constitutive Expression of native IDH1 and IDH1 R132H
  • pcMV3 - Backbone Vector for constitutive Expression of native EGFR plasmids
  • p135 - containing Part for Poly-A-Signal (hGh)
  • p147 - Backbone vector for procaryotic expression of pASTERSIK

Amplification of cloning fragments and sensor sequences

In addition, we design our fragments and sensor sequences needed to achieve our project goals and amplify the fragments with appropriate primer pairs designed through in silico analyzes. After amplification of cloning fragments and sensor sequences gel electrophoresis and Sequencing experiments verified the present of desired sequences:

Figure 2: Amplification of Fragment 1 for ASTERSIK with UTR of HBA1 and HBB


Figure 3: Amplification of Fragment 2 for ASTERSIK


Figure 4: Amplification of Fragment 3 for ASTERSIK with UTR of HBA1 and HBB


Figure 5: Amplification of Fragment IDH1 wild type and mutant.


Table 1: EGFR sensors designed in the first engineering cycle iteration. The two sensors EGFR_CAA5991 and EGFR_CCA7127 were generated using our own software. EGFR_CCA7127 was used as a template for designing two more sensors, EGFR_CCA7127_DS and EGFR_CCA7127_MM, with additional stop-codons and mismatches, respectively. Depicted are the cDNA sequences of the different EGFR sensors hybridized to their respective trigger sequences (portions of the EGFR transcript).
Sensor Features Diagram
EGFR_CAA5991 Targets the CAA 5991 triplet in the 3'UTR. Sensor EGFR_CAA5991
EGFR_CCA7127 Targets the CCA 7127 triplet in the 3'UTR. Sensor EGFR_CCA7127
EGFR_CCA7127_DS Targets the CCA 7127 triplet in the 3'UTR, with an additional stop-codon opposite to a CAA triplet. Sensor EGFR_CCA7127_DS
EGFR_CCA7127_MM Targets the CCA 7127 triplet in the 3'UTR, with 20 mismatching bases introduced. Sensor EGFR_CCA7127_MM

☑ Sensor sequences overlap with in silico expectations
☑ successfully amplification of sensor sequences and insert sequences
However, we tested different experimental setups, conditions, materials and different combinations of various suppliers to improve the efficiency of our downstream experiments for e.g. different polymerases for amplification or restriction enzymes for digestions. All protocols contributed to the iGEM community and the synthetic biology community are optimized to reconstruct our results and implement pASTERISK in their own workflow.

Digestion of backbone plasmids and sequences

As part of the preparation to perform construct assemblies, all isolated vectors were digested using appropriate restriction enzymes. In comparison to the fragments, all linearized vectors were gel electrophoretically purified. Ether gel extraction kit or PCR cleanup kit were used to purify and concentrate the fragments.

  • pSB1C3 - digestion with EcoRI / PstI to cut GFP sequence out of cloning side- length of backbone after digestion
    Figure 6: Double digest of pSB1c3 with EcoRI and PstI.


  • pcDNA5 - digestion with BamHI / NotI to prohibit relegation - length of backbone after digestion
    Figure 7: Double digest of pcDNA5 with BamHI and NotI.


  • p135 - digestion to extract sequence for Poly-A-Signal - length of desired fragment
  • p147 - digestion to extract sequence for CMV promoter - length of desired fragment

* successfully isolated separated PCR products for Part Construction

Construction assemblies and cloning strategies

Using different cloning strategies including gibson assembly, restriction enzyme cloning and biobrick assembly, we successfully integrated our fragments and sensor sequences in the entry site of our backbone plasmids. As an alternative we improve the overlap extension PCR to assembly fragments with appropriate overlap. The lossless integration and correct orientation of the inserts were validated via Colony PCR and Sequencing experiments.

Figure 8: Colony PCR of pASTERISK-HBA1 after assembly.


Figure 9: Colony PCR of pASTERISK-HBB after assembly.


Sequencing Results

☑ All part construction were successfully performed and validated via sequencing experiments

☑ All pASTERISK entry vectors are cloned and ready to use for in vitro transcription

See the mRNA therapy here.