Overview
We provide you with a toolbox for in vivo site-specific continuous mutagenesis. The pivotal elements in the collection are Cre recombinase and Moloney Murine Leukemia Virus reverse transcriptase (MMLV-RT). Our collection contains Cre attached with different degradation tags, and RT under different inducible promoters to achieve diverse steady-state expression level. All parts are BioBrick RFC10 compatible, well characterized, for researchers to choose their desired ones. To initiate reverse transcription and recombination, additional regulatory sequences are required. We provide incompatible loxP sites for Cre and primer binding site along with other reverse transcription completion sequences for RT. We determined our most favored construct based on both the experimental and modeling result. Our collection provides researchers with a set of elements necessary for mutagenesis, and they can assemble the system based on their own needs.
The Reverse Transcriptase Collection
In order to produce the reverse transcriptase effectively inside E. coli cells, we have constructed a series of promoters expressing gag-pol polyprotein with or without operons controlling the transcription. Cloned into a proper protein expression vector, we believe that reverse transcriptase can be expressed in E. coli.
| Part Number | Part Name | Notes |
|---|---|---|
| BBa_K3257106 | gag-Q-pol with T5 Promoter | IPTG Inducible |
| BBa_K3257107 | gag-Q-pol with T7 Promoter | IPTG Inducible |
| BBa_K3257108 | gag-Q-pol with J23119 Promoter | IPTG Inducible |
| BBa_K3257110 | gag-Q-pol with LacUV5 Promoter | IPTG Inducible |
The ChlR Gene Collection
This collection includes wild-type Chloramphinicol resistence gene and its 7 nonsense mutants, which are used as targets of our in vivo sequence-specific toolbox for continuous mutagenesis system. By multiple rounds of mutagenesis process, nonsense mutated ChlR gene will undergo recovery mutation and regain its ability of Chloramphinicol resistence.
| Part Number | Part Name | Notes |
| BBa_K3257111 | ChlR | Wild-type ChlR gene with regulatory elements. |
| BBa_K3257112 | ChlR_T33X | T33X mutant of ChlR gene with regulatory elements. |
| BBa_K3257113 | ChlR_Y92X | Y92X mutant of ChlR gene with regulatory elements. |
| BBa_K3257114 | ChlR_E97X | E97X mutant of ChlR gene with regulatory elements. |
| BBa_K3257115 | ChlR_S121X | S121X mutant of ChlR gene with regulatory elements. |
| BBa_K3257116 | ChlR_L158X | L158X mutant of ChlR gene with regulatory elements. |
| BBa_K3257117 | ChlR_K182X | K182X mutant of ChlR gene with regulatory elements. |
| BBa_K3257118 | ChlR_Q190X | Q190X mutant of ChlR gene with regulatory elements. |
The Reverse Transcription Initiation Collection
This collection includes the components necessary for the reverse transcription initiation, in which tRNAPro and PBS acts as a primer and its binding site, and others are involved in the first and second strand transfer process.
| Part Number | Part Name | Notes |
|---|---|---|
| BBa_K3257060 | PolyPurine Tract(PPT) | Involved in the first strand transfer in reverse transcription process |
| BBa_K3257061 | R Region | Involved in the first strand transfer in reverse transcription process |
| BBa_K3257062 | U5 Region | Important for primer binding and virus encapsidation |
| BBa_K3257063 | Primer Binding Site(PBS) | Providing bingding site for reverse transcriptase |
| BBa_K3257076 | tRNA Primer | Initiating the reverse transcription process |
The Degradation Tag Collection
This collection includes 5 different degradation tags with different degradation rate and steady-state concentration when fused after a CDS of a protein. We have measured the induction curve and growth curve of BL21(DE3) containing EGFP fused with degradation tags (Figure 1).
| Part Number | Part Name | Notes |
|---|---|---|
| BBa_K3257069 | SsrA(LAA) | LAA stands for the last 3 amino acid residues of this degradation tag and this is a widely-used wild-type tag. |
| BBa_K3257070 | SsrA(LAV) | LAV stands for the last 3 amino acid residues of this degradation tag and it is mutated from SsrA(LAA). |
| BBa_K3257071 | SsrA(LVA) | LVA stands for the last 3 amino acid residues of this degradation tag and it is mutated from SsrA(LAA). |
| BBa_K3257072 | SsrA(LVV) | LVA stands for the last 3 amino acid residues of this degradation tag and it is mutated from SsrA(LAA). |
| BBa_K3257073 | SsrA(WVLAA) | WVLAA stands for the last 5 amino acid residues of this degradation tag and it is mutated from SsrA(LAA). |
The Cre Recombinase with Degradation Tags Collection
Combined with Degradation Tags Collection, the leakage of Cre recombinase is solved and difference in expression level between Cre and RT is achieved in an easier way. The steady state concentration of Cre recombinase lowers to a functional level.
| Part Number | Part Name | Notes |
|---|---|---|
| BBa_K3257101 | Cre with SsrA(WVLAA) | Cre recombinase tightly attached with SSr(WVLAA) |
| BBa_K3257102 | Cre with SsrA(LAA) | Cre recombinase tightly attached with SSr(LAA) |
| BBa_K3257103 | Cre with SsrA(LAV) | Cre recombinase tightly attached with SSr(LAV) |
| BBa_K3257104 | Cre with SsrA(LVA) | Cre recombinase tightly attached with SSr(LVA) |
| BBa_K3257105 | Cre with SsrA(LVV) | Cre recombinase tightly attached with SSr(LVV) |
The lox Sites Collection
| Part Number | Part Name | Notes |
|---|---|---|
| BBa_K3257064 | loxP | Wild-type lox site that can be recognized by Cre recombinase. |
| BBa_K3257074 | lox2272 | Orthogonal lox site mutated from loxP. |
| BBa_K3257075 | lox5171 | Orthogonal lox site mutated from loxP. |
This collection includes 4 different types of lox sites. The lox511, lox2272, lox5171 are all orthogonal to loxP as we can see from their inability to be knocked out by Cre if there are loxP on one side and other lox sites on the other side.
After double transformation, colony PCR was done and agarose gel shows that there remains the original DNA fragment.