Team:Hong Kong HKU/Composite Part
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COMPOSITE PARTS
CRISPR-Cas9 contaiment device repressed by arabinose and tryptophan- BBa_K1774000
Overview
This is a prototype containment device which aims to control accidental leakage of GMM once it escape from medium containing arabinose and tryptophan. The device consists of two modules, an arabinose repressible cas9 expression module and a tryptophan repressible gRNA expression module. Since we plan to experiment this device on E. coli BL21 (DE3), we have designed a gRNA that target the chassis only. Under the presence of sufficient amount of arabinose and tryptophan the device remain silent, however when bacteria escape to environment without arabinose and tryptophan, Cas9 and gRNA targeting DNA polymerase III alpha subunit (dnaE) will be expressed. Subsequent cleavage of the targeted sequence may hinder replication of bacteria.
CRISPR-Cas9
Our device utilize CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats, CRISPR-associated), an bacterial adaptive immune system originated from Streptococcus pyogenes SF370 which might play an important role in combating bacteriophages by destroying phage DNA. The wild type system constitute of three essential components. (1) Cas9 endonuclease with two DNA cleavage sites (RuvC and HNH). (2) A non-coding CRISPR array composes of series of repeat-spacer which contain sequence derived from invading DNA. Transcription and processing of the CRISPR array produce number of crRNA carrying 20nt complementary to the target sequence. (3) A trancRNA (trans-activating crRNA) which form a RNA duplex (trancRNA:crRNA) with crRNA that direct binding of Cas9 to target DNA. Base pairing of tracRNA:crRNA with target sequence immediate upstream of protospacer adjacent motif (PAM), in this case 5'-NGG-3' initiates double strand break by RuvC and NHN nuclease domain in Cas9 endonuclease. In order to simplify the device, we have reprogram the system with single guide RNA (sgRNA) instead of using the CRISPR array and tracRNA. This reprogramming is also widely adopted in many genome editing applications. Moreover, this construct also make use of codon optimized Cas9 (BBa_K1774001) for better translation efficiency in E. coli.
How the device work
OFF state -- With arabinose and tryptophan
1. Arabinose activate pBAD by repressing AraC
2. Tryptophan repress pTrp by activating TrpR
3. Activation of pBAD initiate expression of CI which repress expression of Cas9
4. Repression of pTrp lead to low expression of gRNA
5. The expression of CRISPR-Cas9 system is repressed by the presence of arabinose and tryptophan
ON state -- Without arabinose and tryptophan
1. Net consumption of arabinose and tryptophan repress pBAD and activate pTrp
2a. Repression and degradation of CI activate Cas9 expression
2b. Activation of pTrp initiate transcription of gRNA
3. Expression of both Cas9 and gRNA lead to cleavage of dnaE, hence inhibit replication and perhaps lead to cell death
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trpR-pTrp inverter (RBS-trpR-TT-pTrp)- BBa_K1774003
Overview
This is a switch composed of 2 basic parts, trpR, E.coli tryptophan repressor and ptrp, tryptophan promoter and operator. Under the presence of sufficient amount of tryptophan the switch remains silent, however when the environment lacks tryptophan, the expression of the switch in is on. This can be used as a killing switch in synthetic organisms to prevent the escape of bacteria.
How the device work
OFF state -- With trptonphan
1. Tryptophan repress pTrp by activating TrpR
2. Tryptophan co-represses the pTrp
3. The expression is repressed
ON state -- Without tryptophan
1. Absence of tryptophan activate pTrp
2. Activation of pTrp leads to gene expression