Engineering Success

Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.

Introduction

The iGEM Engineering Cycle composes of 4 stages – Design, Build, Test and Learn – each entailing a developmental process towards an ideal biological system. The main processes behind each stage in the iterative process is as follows:

  • Design – Conceptualization and planning; brainstorming possible ideas
  • Build – Build our biological system through molecular cloning techniques
  • Test – Test our biological system and evaluate its efficiency
  • Learn – Reflect on its flaws and improve the system

    • This iterative process allows us to refine our biological system through the mistakes learnt from the previous cycle, thereby building towards an ideal biological system.

    Fig 1: iGEM Engineering Cycle

    Design-Build-Test-Learn

    Our iGEM Engineering Cycle encompasses the developmental process behind our T7RNAP-expressing plasmid. Below shows the 3 iterations, each detailing the features, flaws and improvements to the construction of our plasmid. The aim of the Engineering Cycle was to build a plasmid that was capable of expressing T7 RNA Polymerase (T7RNAP) stably, thereby allowing us to investigate our protein of interest.

    Design

  • Expression of T7RNAP under a constitutive promoter J23104.
  • High-copy number plasmid (pUC) with a strong origin of replication (oriR).
  • Chloramphenicol as the selection marker.

    • Build

    The plasmid was constructed using Gibson assembly involving parts BBa_J364008 (as the backbone) and BBa_J435007 (encoding T7RNAP).

    Fig 2: Design of our initial T7-expressing plasmid - plasmid 1

    Test

    Despite 2 separate rounds of cloning the desired plasmid, we were unable to isolate our plasmid of interest with the correct sequence.

  • In the first round of cloning, our T7 expression cassette was missing several expression elements (ie. J23104 promoter, RBS and terminator) to properly express the T7RNAP. The defects observed in these clones were despite the fact that the primers used and original plasmids have already contained these elements.
    • Fig 3: Identified missing expression elements in Plasmid 1 after Sanger sequencing. [LEFT] Missing constitutive promoter J23104 and RBS. [RIGHT] Missing rrnB T1 terminator and T7Te terminator

  • In the second round of cloning, new primers were designed and utilised for cloning. However, we still observed either a truncated protein or a deletion of part of the T7RNAP sequence in several of the colonies sequences.
    • Fig 4: Identified missing protein sequences in Plasmid 1 after Sanger sequencing. [LEFT] Missing 'T' in the first start codon ATG, resulting in the expression of a truncated protein variant. Whilst not shown, the truncated variant was still capable of transcription. [RIGHT] Deletion of the entire T7RNAP coding sequence; resulting in only the immediate N- and C-terminus amino acid residues

    Learn

    The defects observed from both rounds of cloning seems to predominantly lie with the issue of stably expressing the T7 RNAP within its host; which ties in which the issue of expression toxicity and lethality threshold.

    Conventionally, T7RNAP is used in in vivo protein expression (eg. BL21 (DE3) or other DE3 counterparts) owing to its specific promoter recognition and high transcriptional activity1. In these strains, T7RNAP is commonly expressed under an inducible promoter, to regulate the expression and allow the temporal control of T7RNAP (ie. induce only when required). This temporal control of T7RNAP has several benefits, with the most obvious linking to the fitness of the host.

    T7RNAP is reported to have 8-fold higher transcriptional activity than host bacterial RNAPs2. As such, when cloning the T7RNAP under a constitutive promoter, we hypothesis the high transcriptional activity of T7RNAP to eventually become toxic to its host, When such a strong RNA polymerase is constitutively expressed in a host, it lowers the fitness of the host due to inefficient allocation of metabolic resources. This forces the host to negatively select variants through undesirable mutations (usually with worse-performing traits) to better counteract the original strong RNA polymerase activity3. This was what we observed where the cloning of T7RNAP under a constitutive promoter had several setbacks.

    As such, to better regulate the expression of T7RNAP and mitigate the toxicity associated, we designed the constitutive promoter to be under the regulation of a lac operon, a system commonly used in commercial DE3 bacterial strains.

    Design

  • Expression of T7RNAP under a regulated promoter; constitutive promoter J23104 is upstream of and regulated by a lac operator.
  • Addition of lacI repressor to aid in the regulation of the promoter-lac operator.

    • Build

    The plasmid was constructed using Gibson assembly involving parts BBa_J364008 (as the backbone) and BBa_J435007 (T7RNAP). LacI sequence with promoter was derived from plasmid Addgene #65767.

    Fig 5: Design of our second T7-expressing plasmid - plasmid 1a

    Test

    The cloning of plasmid 1a was relatively easier, with the various expression elements and the sequence of the protein still intact. The addition of regulatory elements shows promise in regulating the expression of T7RNAP. The plasmid (plasmid 1a) was subsequently transformed into competent E. coli reporter cells already containing our reporter plasmid, and plated onto double antibiotic (Kan + CmR) LB agar plates. Colony PCR was then performed to determine if both plasmids were retained within the E. coli reporter cells. Whilst the colony PCR results show that both plasmids were retained, it also showed an increase in the band size of the fragment corresponding to the T7RNAP, possibly due to an insertion sequence.

    Fig 6: Colony PCR results after transforming plasmid 1a into competent E.coli reporter cells. [LEFT] Whilst the band of interest (expected size: ~800bp) corresponding to our reporter casette in plasmid C3 was correct, the band of interest corresponding (observed to be either ~2000bp or ~3000bp) to plasmid 1a was observed to be different from the expected size of 1200bp. [RIGHT] Colony PCR was repeated using untransformed plasmids as control. Only the band of interest derived from the control samples showed the expected band size of 1200bp.

    Transformation was thus performed again, with plating onto two different antibiotic agar plates, as well as the addition of a control. Colony PCR was also performed again to observe for any similar pattern of insertion sequences, and if the insertion was harboured near the N-terminus or C-terminus of the T7RNAP. Our results support the previous observation where there was an insertion sequence within the open reading frame of the T7RNAP coding sequence, with insertion sequence found nearer to the N-terminus. The frequency of these insertion sequences was also observed to be higher in our reporter cells.

  • [1] - Plasmid 1a transformed into competent E. coli reporter cells and plated onto Kan + CmR LB agar plates
  • [2] - Plasmid 1a transformed into competent E. coli reporter cells and plated onto CmR LB agar plates
  • [3] - Plasmid 1a transformed into competent empty E. coli cells and plated onto CmR LB agar plates

    • Fig 7: Colony PCR was performed again after transformation of plasmid 1a into competent E. coli reporter and empty cells. An increase in band size was observed in the fragment corresponding to the T7RNAP sequence. The colony PCR results showed that the insertion sequence is consistently found closer to the N-terminus coding region of the protein. Box in red are colonies that were further cultured for plasmid purification.

    In order to determine the identity of the insertion sequences, colonies that were boxed in red were further cultured to perform plasmid purification (miniprep). Plasmids were isolated and sent for Sanger sequencing to reveal the identity of these insertion sequences. Sanger sequencing results from [2]-4 support the observation that the increase in band size was due to an insertion sequence (identified as IS1 by Snapgene) within the coding region (near the N-terminus of T7RNAP). The location of the insertion was also highly specific, between amino acids 315 and 317.

    Fig 8: Sanger sequencing results of plasmid 1a purified from [2]-4. The insertion sequence was identified as IS1, and was inserted before amino acid positions 315 - 317.

    Learn

    The insertion sequence (IS1) within the coding sequence of T7RNAP was hypothesized to be a "defence mechanism" from the Stbl3 E.coli cells after transformation. This observation was also more commonly observed when transformed with competent cells already containing the reporter plasmid. It was possible that the IS1 sequence acts to “break” the coding sequence of T7RNAP, thereby knocking out the expression of the T7RNAP. Overall, while the plasmid was clone-able, our test results suggest that the expression of T7RNAP from plasmid 1a is possibly still toxic and unstable despite the additional regulatory elements such as the lac operator (lacO) and lacI repressor. This suggests that the current regulatory elements are still insufficient to control the expression of T7RNAP.

    To further regulate and maintain a stable expression of T7RNAP, an antisense oligonucleotide (ASO) system and a lower-copy number plasmid system was adopted – plasmid 1c.

    Design

  • Addition of an antisense oligonucleotide (ASO) with Hfq scaffold sequence to promote the knockdown of T7RNAP expression4. The ASO aims to targets the +4 to +27 of the coding sequence to knockdown the expression of T7RNAP.
  • Medium-copy number plasmid using a pBR322 backbone

    • Build

    The plasmid was built on from plasmid 1a; using Gibson assembly to add in the antisense oligonucleotide (ASO) system and swap the oriR. ASO was ordered as a gBlock and different oriR was derived from BBa_J428341 (medium-copy pBR322 plasmid).

    Fig 9: Design of our third T7-expressing plasmid - plasmid 1c

    Test

    The cloning of plasmid 1c was swiftly completed, with its sequence verified. Previous regulatory elements (ie. lacO and lacI) were retained, and the ASO system was also included. Similarly, plasmid 1c was subsequently transformed into our competent E. coli reporter cells for an initial screening. Transformed cells were then plated across three different LB agar plates - CmR only, Kan only, and CmR + Kan.

    Fig 10: Colonies were observed for all three different LB agar plates after transformation.

    8 colonies from each plate were then selected for colony PCR; to determine if the respective plasmids were retained within the E. coli cells. Colony PCR results indicate that both the T7-expressing plasmid (CmR resistance marker) and reporter plasmid (Kan resistance marker) were retained on all 8 colonies on the double antibiotic plate (CmR + Kan). These colonies were inoculated and further subcultured. Fluorescence was subsequently observed in only colony 6 after a period of incubation (initially grown at 37°C overnight; then kept at 4°C for 72 hours).

    Fig 11: 8 colonies per plate were selected for colony PCR. Colonies derived from plates on single antibiotic only contained a single plasmid (their corresponding markers). Colonies derived from double antibiotic plate (CmR + Kan) contained both plasmids, albeit with different fragment sizes for the T7RNAP (different sizes are possibly due to deletion or insertion of sequences within the coding region of T7RNAp; however, this was not further pursued due to the interest of time).
    Fig 12: Fluorescence was observed in only colony 6 after bacterial cells were pelleted after incubation. Initial results suggest the reporter system is functional, and that T7RNAP can be stably expressed. Observation of fluorescence is also in line with colony PCR results. [TOP/a] Observation under blue-light. [BOTTOM/b] Observation under physical light.

    In order to determine if the expression of T7RNAP from plasmid 1c is indeed more stable than that of plasmid 1a, we transformed our E. coli reporter cells again with respective plasmids, and performed colony PCR to identify for the presence of any possible insertion sequences within the coding sequence of the T7RNAP. The same pair of primers were used to amplifiy the sequence of interest for both plasmids.

  • Expected band size for plasmid 1a: 1892bp
  • Expected band size for plasmid 1c: 2038bp; due to additional ASO system

    • Fig 13: Colony PCR results showed the presence of insertion sequences within the coding sequence of T7RNAP for E.coli reporter cells transformed with plasmid 1a. Band size corresponding to ~3000bp (different from expected band size of 1892bp) was observed for all 8 colonies picked from reporter cells transformed with plasmid 1a, thereby showing the unstable expression of T7RNAP.

    Learn and Further Actions

    Through our Engineering Cycle, we were able to refine the expression of T7RNAP, a protein that was initially unstable. A combination of various regulatory elements work hand-in-hand together, aimed at knocking the expression levels of T7RNAP down to stable levels. Our last iteration also capitalizes on an ASO system, a system that is not commonly used for plasmid-expressed proteins. Overall, the utilisation of the different regulatory elements allowed us to stably express the wild-type T7RNAP, thereby allowing us to investigate the efficiency of other variants in our random mutagenesis library.

    In addition, given the scope of our project to also explore ancestral sequences, the ASO system can be further refined to regulate the expression of these T7RNAP variants that have low sequence homology.

    References

    1 CHAMBERLIN, M., MCGRATH, J., & WASKELL, L. (1970). New RNA Polymerase from Escherichia coli infected with Bacteriophage T7. Nature (London), 228(5268), 227–231. https://doi.org/10.1038/228227a0

    2 Miroux, B., & Walker, J. E. (1996). Over-production of Proteins inEscherichia coli: Mutant Hosts that Allow Synthesis of some Membrane Proteins and Globular Proteins at High Levels. Journal of Molecular Biology, 260(3), 289–298. https://doi.org/10.1006/jmbi.1996.0399

    3 Kar, S., & Ellington, A. D. (2018). Construction of synthetic T7 RNA polymerase expression systems. , 143, 110–120. https://doi.org/10.1016/j.ymeth.2018.02.022

    4 Liang, X., Li, C., Wang, W., & Li, Q. (2018). Integrating T7 RNA Polymerase and Its Cognate Transcriptional Units for a Host-Independent and Stable Expression System in Single Plasmid. ACS Synthetic Biology, 7(5), 1424–1435. https://doi.org/10.1021/acssynbio.8b00055