gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
pCS Bacterial Recombinant Protein Vector
The pCS vector system is an efficient system for expressing recombinant proteins in E. coli at low temperatures, typically 15°C. It is based on the E. coli cold-shock gene cspA, and also includes part of the lac operon regulatory system. It is considered to be complementary to the pET and pBAD systems that are typically used to express recombinant proteins at 37°C.
Temperature can have a significant impact on the folding of recombinant proteins, which in turn affects protein solubility and stability. As such, many proteins that exhibit poor solubility or stability at 37°C show improved properties at lower temperatures. When a recombinant protein is difficult to produce in pET or pBAD at 37°C, the pCS system could be a useful alternative to try.
The gene of interest is placed into the pCS vector downstream of the cspA promoter, lac operator (LacO), and the 5’ UTR of cspA. A short translation enhancing element (TEE) from the N-terminus of cspA is also included.
Transcription from the cspA promoter is very inefficient at 37°C, because of instability of the 5’ UTR at this temperature, but when the host bacteria is shifted to 15°C, the 5’ UTR adopts a highly stable secondary structure. The TEE sequence enhances translation initiation of the gene of interest through a ribosome trapping process.
The plasmid also carries the natural promoter and coding sequence for LacI. The LacI protein acts on the LacO sequence, adjacent to the cspA promoter, to block transcription of the gene of interest. In the absence of IPTG, this inhibition prevents any leaky expression of the downstream gene. Addition of IPTG blocks the inhibitory action of LacI. This, coupled with shifting of the culture from 37°C to 15°C, can efficiently induce gene expression.
For further information about this vector system, please refer to the papers below.
|Nat Biotechnol. 22:877 (2004)||Cold-shock induced high-yield protein production in E. coli|
|J. Biol. Chem. 274:10079-85||TEE sequence translation enhancement|
E. coli containing the pCS vector is maintained in growth medium lacking IPTG at 37°C. Expression of the gene of interest can be induced by the addition of IPTG to remove LacI-based repression, combined with a temperature shift to 15°C, to activate the cspA promoter and stabilize the cspA 5’-UTR. This system can be used as an alternative when the recombinant protein of interest cannot be produced using the pET or pBAD systems. It is also recommended for expression of cold-stable proteins or proteins which are unstable at 37°C.
Tightly controlled expression: Expression from pCS vectors is strongly repressed at 37°C in the absence of IPTG, and induced by the addition of IPTG and a temperature shift to 15°C.
Expression at low temperatures: The pCS vector system allows expression of recombinant proteins at low temperatures. This may be advantageous for many proteins of interest that cannot be efficiently achieved with other bacterial expression systems.
Host requirements: Unlike pET or pBAD vectors, which require specialized E. coli host strains for recombinant protein expression, pCS can be used to produce recombinant proteins in the same E. coli strain that is used for cloning (typically Stbl3), though the BL21 strain is recommended as the host for protein expression as this strain is engineered to reduce protein degradation.
Sub-maximal expression: pCS vectors are generally not capable of achieving the very high level of expression that is possible with pET vectors.
cspA promoter: Promoter of the cold-shock gene cspA. It drives transcription of the gene of interest at 15°C.
LacO: Binding site for LacI. This element inhibits activity of the cspA promoter when LacI protein is present, preventing leaky expression of the gene of interest.
cspA 5’ UTR: 5’ untranslated region of the cspA gene, which destabilizes the mRNA at 37°C and becomes highly stable at 15°C.
TEE: Translation enhancing element. This sequence is preferentially bound by ribosomes initiating translation, so once bound to the TEE, ribosomes are rarely available to translate other mRNAs.
ORF: The open reading frame of your gene of interest is placed here.
cspA 3’ UTR: 3’ untranslated region of the cspA gene.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.
LacI: The E. coli natural promoter and coding sequence for the lac repressor. Without IPTG present, the LacI protein represses transcription of the gene of interest from the cspA promoter due to the LacO site adjacent to the cspA promoter.