Part:BBa_K5124042

IPTG inducible T7 promoter system

Usage and Biology

The Exeter iGEM 2024 team are designing a rapid detection system for Bovine Tuberculosis (bTB) using CRISPR-Cas detection systems. The literature suggests that bTB infection in cattle can be detected by nucleic acid biomarkers in both blood [1] and tissue samples [2]. Therefore, there was potential to develop tests looking for both DNA and RNA biomarkers in infected cattle.

For our project we needed to express both Lachnospiraceae bacterium ND2006 Cas12a and Leptotrichia wadei Cas13a enzymes at high levels, but we also wanted to control when our enzymes would be expressed. Our supervisor has previously used the IPTG inducible promoter system in the Novagen pET plasmids for over expression of proteins. This includes the T7 bacteriophage promoter in combination with the E. coli lac operator sequence and the strong RBS from T7 bacteriophage gene 10. Transcription in the pET plasmids is terminated by the transcription terminator for bacteriophage T7 RNA polymerase. There are many versions of these sequences in the Registry of Standard Biological Parts but the ones most similar to the pET system are:
• BBa_R0085- T7 promoter sequence
• BBa_K4673024 - E. coli Lac operator
• BBa_K3257011- T7 gene 10 RBS
• BBa_K395601- T7 RNA polymerase terminator

Design and Characterisation

For expression of our enzymes, a composite part comprising R0085, K4673024 and K3257011 with Type IIS prefix and suffix sequences was ordered as a gBlock from IDT. K395601 was also ordered as a gBlock from IDT with Type IIS prefix and suffix sequences. These were cloned into a medium copy plasmid (origin of replication from pBR322 [3]) carrying an ampicillin selection marker with either the coding sequence for LbCas12a (BBa_K5124000) or LwCas13a (BBa_K5124001) using Type IIS cloning.

The resulting expression plasmids were transformed into E. coli BL21(DE3) (Novagen) and protein expression induced by autoinduction media [4]. The enzymes were purified via Ni-affinity and size exclusion chromatography. Please see our Wiki for the detailed protocol (Wiki).

Results

Both enzymes were successfully expressed and purified as verified by SDS-PAGE and Western Blot analysis (Figure 1 and 2). For further results please see BBa_K5124000 or BBa_K5124001.

Figure 1: Cas 12a SDS-PAGE and Western Blot results through the purification process

Figure 2: Cas 13a SDS-PAGE and Western Blot results through the purification process

Figure 3: A - SDS-PAGE showing lanes after the His-Trap and SEC. B - graph showing percentage of Cas 13a protein after the His-Trap and after the SEC. There is a significant increase in the percentage of Cas13a in the overall protein content.

Cas13a proteins were purified from crude cell lysate using His-Trap (Cytiva) columns, and AKTA Pure (Superdex 200 10/300 120mL Cytiva) Size Exclusion Chromatography (SEC). The resulting SDS-Page gel was photographed and analysed to determine the success of purification following each technique. To quantify purity of Cas13a, the following analysis was used:
The image was cropped to the relevant lanes, (shown in yellow on Figure 3a) with the band containing the Cas13a protein also separately cropped (shown in red on Figure 3a).
These images were saved as .png files named img1-4.
A custom Matlab script (on our wiki) was used to import each image, convert to a numerical matrix of RGB intensities, and count blue pixels given a certain threshold (Red <=100, Green <=100, Blue >= 100).
Final values were plotted (Figure 3b)

Conclusions

These results show that this inducible T7 promoter system can be used to express multiple proteins (both Cas13a and Cas12a), to a usable concentration, making it a useful tool for teams in the future to use, if they wanted to express Cas12a, Cas13a, or any other protein using induction.

References

1. McLoughlin KE, Correia CN, Browne JA, Magee DA, Nalpas NC, Rue-Albrecht K, et al. RNA-Seq Transcriptome Analysis of Peripheral Blood From Cattle Infected With Mycobacterium bovis Across an Experimental Time Course. Frontiers in Veterinary Science. 2021; 8:662002.

2. Taylor GM, Worth DR, Palmer S, Jahans K, Hewinson RG. Rapid detection of Mycobacterium bovis DNA in cattle lymph nodes with visible lesions using PCR. BMC Vet Res. 2007 Jun 13; 3:12.

3. Sutcliffe JG. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979; 43 Pt 1:77-90.

4. Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005 May; 41(1):207-34.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]