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Cloning

Abstract

To express the various components of our scaffold for protein conjugation and assembly experiments, our DNA constructs were cloned into appropriate plasmid vectors. We used Gibson assembly cloning to assemble the DNA inserts with the vectors in one simple isothermal reaction¹. The products of the reaction were transformed into competent DH5-alpha Escherichia coli cells and colonies were screened for recombinant plasmids. Plasmids containing the desired DNA inserts were transformed into T7 cells for protein expression and purification experiments. 8 constructs were successfully cloned into corresponding pET-Duet1 and pRSF-Duet1 plasmids, while 6 constructs were cloned into pET-19b plasmids.

Introduction

The components of the prefoldin scaffold and the associated attached enzymes were to be expressed for enzymatic and self-assembly experiments. To produce the proteins required for these analytical experiments, our gene of interests were to be cloned into an appropriate plasmid vector for expression and purification in E. coli cells. This page describes the methods undertaken to produce recombinant plasmids containing our DNA constructs for protein expression. 8 codon optimised DNA constructs were designed and synthesised in the form of g-Blocks from Integrated DNA Technologies (IDT).

The hetero-hexameric structure of our scaffold is composed of two alpha prefoldin (aPFD) subunits and four beta prefoldin (bPFD) subunits covalently attached to enzymes. ² To produce a functional and complete scaffold, genes encoding the proteins which form the scaffold must first be cloned into an appropriate vector. Plasmids containing aPFD and bPFD were constructed. In addition, aPFD-SpyCatcher and bPFD-SnoopCatcher fusion constructs were created for comparative enzyme activity experiments.

SpyTags ³and SnoopTags ³ were fused to two enzymes, indole-3-acetamide hydrolase (IaaH) and tryptophan 2-monoxygenase (IaaM) and cloned into appropriate plasmid vectors. Expression of these plasmids would enable protein conjugation experiments with the prefoldin-catcher protein constructs. Fluorescent mCerulean3-SnoopTag and mVenus-SpyTag gBlocks were also cloned into plasmids for Försters Resonance Energy Transfer (FRET) experiments. 6x His-Tags were also attached to the start of each DNA construct via a GSG linker, which would allow the recovery of purified protein.

All original gBlock designs contain the BioBrick prefix and suffix sequences which were flanked by 20-25 bp long 5’ and 3’ Gibson overhangs. Cloning the BioBrick restriction sites into the DNA construct allowed excision of the insert out from the plasmid vector for diagnostic purposes and for transfer of the inserts into pSB1C3. Gibson Assembly cloning techniques were used to clone the DNA constructs into the plasmids. 5’ exonuclease activity generates complementary overhang sequences on the insert and vector, and polymerase fills in the gaps of the single strand regions. DNA ligase seals the nicks of the gaps, allowing the two fragments to covalently link together (Figure 1) ¹.

Figure 1: Diagram illustrating the process of Gibson assembly sequence insertion into the plasmid vector ¹.

Our DNA constructs were cloned into pETDuet-1 and pRSFDuet-1 plasmid vectors, as well as pET-19b in our later experiments. The Duet vectors carry two expression units that are controlled by a T7-lac promoter and terminator for protein expression. The Duet plasmids, pETDuet-1 and pET-19b, both possess an ampicillin resistance gene (Figure 2) while pRSFDuet-1 confers kanamycin resistance. These specific vectors were chosen so that the prefoldin-catcher and enzyme-tag DNA constructs could be cloned into the same cell, allowing the entire scaffold to be expressed simultaneously. Furthermore, pETDuet-1 and pRSFDuet-1 plasmids possess different origins of replication, which enables in vivo production of the scaffold-enzyme complex.

Figure 2: Plasmid maps depicting pETDuet-1, pRSFDuet-1 and pET19-b. Resistance genes are shown in red. Images were generated by Benchling.

8 DNA constructs were successfully cloned into pET-Duet1 and pRSF-Duet1 plasmids. However, these plasmids were not able to be expressed. The constructs were then redesigned to omit the iGEM prefix and suffix sequences and the Gibson overhangs were modified. This enabled the cloning of 6 modified constructs into the pET-19b vector for protein expression and purification.

Table 1: The specific plasmid vector the DNA constructs were to be cloned into. Circular pETDuet-1 and pRSFDuet-1 plasmids and linearised pET-19b were kindly supplied by Dr Dominic Glover.

pETDuet-1	pRSFDuet-1	pET-19b
aPFD	IaaH-SpyTag	aPFD
bPFD	IaaM-SnoopTag	bPFD
aPFD-SpyCatcher	mCerulean3-SnoopTag	IaaH-SpyTag
bPFD-SnoopCatcher	mVenus-SpyTag	IaaM-SnoopTag
		IaaH
		IaaM

Aim

To clone genes encoding the parts required to form our scaffold into appropriate plasmid vectors by Gibson assembly, involving:

1. Cloning original DNA constructs into pETDuet-1 and pRSFDuet-1
2. Cloning modified DNA constructs into pET-19b

DNA design

Figure 3: 8 DNA constructs designed by Brian Ee. Images were generated by Benchling.

8 gBlocks were designed for cloning by Gibson assembly into the first multiple cloning sites of pETDuet-1 and pRSFDuet-1. All sequences were designed with an N-terminal 6xHis-Tag immediately after the start codon to enable purification using Nickel affinity, followed by a Glycine-Serine-Glycine (GSG) linker. The GSG linker provides flexibility as the side chains of glycine and serine are small, and can allow the 6xHis-Tag to move freely in solution. The amino acid sequences for each protein were obtained. For fusion proteins, a GSGSGSGSG linker and SpyCatcher or SnoopCatcher then followed, or a GSG linker and SpyTag or SnoopTag. A longer 9 amino acid linker is used for SpyCatcher and SnoopCatcher fusion proteins as the catcher domains are large and may sterically interfere with protein folding if the C-terminus of the original protein is not solvent accessible. An increased linker length on the aPFD-SpyCatcher and bPFD-SnoopCatcher fusions may also enable the scaffold to accommodate the attachment of large enzymes. The DNA sequence was obtained by codon optimisation for E. coliwith manual removal of EcoRI, XbaI, SpeI and PstI restriction sites for RFC10 compatibility. The BioBrick prefix and suffix were then placed on the 5’ and 3’ ends of each sequence. Finally, the DNA sequences were flanked with 25 bp Gibson overhangs identical to the 25 bp immediately upstream and downstream of the insertion site into pETDuet-1 and pRSFDuet-1.

Method

pETDuet-1 and pRSFDuet-1 plasmids were linearised with PCR, removing the first multiple cloning site. Enzymes were removed by PCR clean up (Sigma Aldrich). A DpnI digest was performed to remove template circular plasmid and cleaned up again. Linearity was confirmed by agarose gel electrophoresis. Gibson assembly was used to construct plasmids by combining linearised plasmids with gBlocks ordered from IDT. The Gibson assembly product was heat shocked transformed into E. coli DH5-alpha (NEB) cells and plated onto Luria broth media plates with the appropriate antibiotic. A colony PCR was performed to identify single colonies that had been successfully transformed with Gibson assembly products and analysed with agarose gel electrophoresis. Successful transformants were grown in 10 mL of LB with appropriate antibiotic and plasmids were prepared using QIAprep Spin Miniprep Kit (Qiagen). To confirm the insert of our DNA construct, the plasmids were digested with restriction enzymes that excised the insert and analysed with agarose gel electrophoresis. Sanger sequencing was performed by the Ramaciotti Centre for Genomics to verify gene sequences.

For the transfer of inserts into pET-19b, primers were designed to PCR amplify the inserts that remove the BioBrick prefix and suffix, and add Gibson overhangs appropriate for insertion into the multiple cloning site of pET-19b. Gibson assembly was then performed as previously described with PCR linearised pET-19b.

Figure 4: Flowchart depicting the overall cloning process undertaken.

Click here for detailed cloning protocols!

Results

Cloning original DNA constructs into pETDuet-1 and pRSFDuet-1

All 8 original DNA constructs were successfully cloned into pET-Duet1 and pRSF-Duet1 plasmid vectors. Figure 5 displays the gel electrophoresis results of a plasmid digest with EcoRI and PstI, depicting the presence of the target insert gene. These recombinant plasmids were used for protein expression and purification experiments.

Figure 5: 1% agarose gel electrophoresis demonstrating the construction of plasmids containing the desired DNA inserts. Plasmids were restriction enzyme digested with EcoRI and PstI and analysed by agarose gel electrophoresis. The red boxes indicate the presence of the insert at the expected size in comparison to the 2-Log DNA marker.

Cloning modified DNA constructs into pET-19b

The 8 recombinant pETDuet-1 and pRSFDuet-1 plasmid vectors were unable to be expressed, so the DNA constructs were modified and cloned into the pET-19b vector instead. Figure 6 displays the results of the EcoRI and XbaI restriction enzyme digest on the aPFD, bPFD and IaaH-SpyTag recombinant plasmids. The following modified constructs were successfully cloned into pET-19b:

• aPFD
• bPFD
• aPFD-SpyCatcher
• bPFD-SnoopCatcher
• IaaH-SpyTag
• IaaM-SnoopTag

Figure 6: 1% agarose gel electrophoresis demonstrating the construction of plasmids containing the desired DNA inserts. Plasmids were restriction enzyme digested with EcoRI and XbaI and analysed by agarose gel electrophoresis. The red boxes indicate the presence of the insert at the expected size in comparison to the 2-Log DNA marker.

The following BioBricks from the provided distribution plates were cloned into pET-19b for BioBrick part experiments for the creation of our Improved Part.

• BBa_K1789000: IaaM
• BBa_K1789001: IaaH

Discussion

The initial aim of cloning all 8 DNA constructs into pETDuet-1 and pRSFDuet-1 plasmid vectors was successful. We originally decided to utilise these two Duet vectors as it would enable us to co-transform and express two target genes into MCS1 and MCS2 if time permitted. This would streamline the process in which our hetero-hexameric complex assembles, increasing the efficiency of future cloning experiments. However, we were unable to express our target proteins when the recombinant plasmids were transformed into expression strains for protein purification experiments. We hypothesised that this was due to the 20 bp long BioBrick prefix situated between the ribosomal binding site (RBS) and the start codon of our construct. This displaces the RBS away from the start of transcription, which is the likely cause for the difficulties experienced with protein expression. Translation studies in E. coli have demonstrated that the optimal spacing between the RBS and the start codon in E. coli ranges from 7-9 nucleotides ⁴.

We decided to clone the DNA constructs into the pET-19b vector as it was used successfully in previous cloning experiments for our collaborators. Dr Dominic Glover kindly supplied us with linearised pET-19b plasmids for our experiments, and we were successful in cloning 6 of our 8 DNA constructs into these vectors. Unfortunately due to time restrictions, we were unable to clone all 8 DNA constructs in pET-19b. In the future, we hope to clone more parts into pET-19b which would enable us to perform more assembly tests and ultimately piece together and characterise a complete and functional scaffold.

References

1. Gibson, D. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6, 343-345 (2009).
2. Siegert, R., Leroux, M., Scheufler, C., Hartl, F. & Moarefi, I. Structure of the Molecular Chaperone Prefoldin. Cell 103, 621-632 (2000).
3. Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences 109, E690-E697 (2012).
4. Vellanoweth, R. & Rabinowitz, J. The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. Molecular Microbiology 6, 1105-1114 (1992).