Part:BBa_K4673024
The repressor binds to the operator
Within many prokaryotic bacterial structures, the operator serves as the primary region of DNA where the regulatory molecules of an operon are attached. The operator doesn’t exist in eukaryotes. Instead, it is the transcription factors that control the interactions associated with promoter portions.
In E. coli strains, the lac operator segment is generally characterized as a portion situated between the lac operon and the lac promoter, and it interacts with the repressor molecule(lacI). On DNA sequences, the lac repressor(lacI) specifically binds to the lac operator, inhibiting the expression of the lac operon. The binding also prevents the RNA polymerase from initiating transcription for the genes situated downstream of the lac operator since the lac operator overlaps the lac promoter. Consequently, the primary job of the lac operator is to regulate the process of gene expression by facilitating the binding of the operon and the regulatory molecules.
As a part of the pET-22b-LK vector in our team’s project, the lac operator functions as the binding region for the lacI repressor, playing an important role in the IPTG induction mechanism. The expression of lac operon occurs only when lactose is present and glucose is absent. The structure of the lac operon comprises the lac operator, which is a negative regulatory overlap with the promoter. Therefore, when lac repressor binds to lac operator, RNA polymerase cannot effectively attach to the promoter and start transcription.
Exeter Team 2024
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
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 (see BBa_K5124042). 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).
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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