Part:BBa_K3333013
lacI -- tac promoter -- λ-red -- lambda tL3 terminator
This is a composite part can be divided of the lacI gene, the tac promoter, lambda-red and a downstream transcriptional terminator (Lambda tL3 terminator).Lambda-Red is a recombinase system, which originated from the bacteriophage λ. This part can mediate recombination for multiplex genome editing.
Usage and Biology
The lactose operon (lac operon) is an operon required for the transport and metabolism of lactose. And it consists of 3 structural genes, and a promoter, a terminator, regulator, and an operator. The three structural genes are: lacZ, lacY, and lacA. Normally, the lac operon is turned off. A repressor protein binds the operator region upstream of the operon preventing transcription. When lactose is not available, the lac repressor binds tightly to the operator, preventing transcription by RNA polymerase. However, when lactose is present, the lac repressor loses its ability to bind DNA. It floats off the operator, clearing the way for RNA polymerase to transcribe the operon. At the meantime, catabolite activator protein (CAP) binds to a region of DNA just before the lac operon promoter and helps RNA polymerase attach to the promoter, driving high levels of transcription. And research has shown that the promoter of the Iac1 is active and the lac repressor functional in Pseudomonas[1].
The tac promoter is a functional hybrid derived from the trp and lac promoters. Research has shown that tac promoters can be repressed by the lac repressor[1]. The tac promoter appeared to be at least 10 times more efficient than the lac UV5 promoter and at least 3 times as strong as the fully trp promoter[2]. At last hybrid tac promoter and the promoter of the lac1 gene of Escherichia coli are active in Pseudomonas[1].
Lambda-red includes three genes, γ, β, and exo, these genes respectively encode protein Gam, Bet, and Exo [3]. λ-red system is a widely used recombination system deriving from phage λ, containing three proteins: Exo, Beta and Gam. This system can help recombine dsDNA/ssDNA into a new DNA molecule, serving as a useful and practical tool in many other projects. There are currently three models explaining the elaborate mechanisms of the λ-red system: RecA-dependent, ssDNA annealing (RecA-independent) and the recent Replisome Invasion/Template Switch model[4]. What makes the λ-red system stands out is its high efficiency compared to restriction enzymes as well as its ability to promote recombination events between DNA species with as little as 40bp of shared sequence at high frequency.[5] It is also worth mentioning that recombinant formation promoted by the λ-red system is IPTG dependent, supported by the fact that longer exposure to IPTG results in greater amounts of recombinants per competent cell.[6]
λ Exo, working as an exonuclease with a preference for 5’-phosphates, degrades the 5’-ended strand dsDNA and thus creating a 3’-ended overhang. This exonuclease exists as a trimer in a toroidal shape with a funnel-shaped central channel. It is presumed that the dsDNA passes through the central channel and is then acted upon the by one of the three acting sites and finally exits as ssDNA.[4] Particularly, the side chain of Leu78 inserts between the second and third bases of the 5’-ended strand so as to separate the two terminal nucleotides in the active site.[7] Besides, in the active site to which the DNA is bound, there are two Mg2+ ions showing octahedral coordination, revealing that λ exonuclease uses a classic two-metal mechanism which is also seen in several TypeⅡrestriction endonucleases.[7]
λ Beta is a protein capable of meditating strand annealing and promoting the recombination of complementary strands. Beta exists in three structural states: small rings, large rings and helical filaments. When encountered with DNA, large beta rings bind ssDNA and therefore initiate the annealing with a complementary strand. The annealing process continues spontaneously to generate a dsDNA supercoiled within the β helical filament [8]. Beta binds to ssDNA and delivers ssDNA to the target replication fork, the Bet bound ssDNA behaves like Okazaki fragment and is introduced into the genome[3]
λ Gam, also known as the anti-RecBCD protein, functions by directing binding to the DNA-binding site of RecBCD and subsequently inhibiting RecBCD’s capability to bind to dsDNA ends. This protein is an all helical dimeric structure with two hydrophobic N-terminal H1 helices sticking out from a dimerization domain. The Gam dimerization domain is proposed to be a double-stranded DNA mimetic while helix H1 may mimic ssDNA. [9] These two helices are speculated to be inserted into channels within the RecB and RecC subunits which were originally occupied by 3’-ssDNA and 5’-ssDNA of an unwound dsDNA substrates. Besides, aromatic residues are assumed to interact with the bases of the ssDNA.[4]
The Lambda tL3 terminator of phage Lambda is grossly rho-independent[10]. the major leftward operon of phage Lambda contains several other terminators and they include the tL3. It is also active in vitro, both in the presence or absence of the rho factor[11]. The tL3 terminator has several features common to other rho-independent termination sequences, including an high G+C region of 2*4-bp symmetry with a 5-bp intervening “loop” and other hairpin structure. At last, Lambda tL3 terminator is about 95% efficient at 30 °C[10].
In order to produce the engineered phages, we constructed two plasmids, one carrying Cas9 and Lambda-Red, while the other one carrying the TA system genes. Cas9 system and Lambda-Red system is associated to edit the natural phages vB_PaeM_SCUT-S1, to make it carry the toxin gene. We are hoping to utilize this λ-red system to insert a toxin gene into the phage genome together with the CRISPR/Cas9 system. As a result, this phage will acquire the ability to cause excessive toxin expression in the host cell after infection.
Experimental approaches
In order to test the effect of Lambda-Red system on phage genome editing, we designed two pairs of homologous arms, which are linked to the relE-tat promoter respectively. Under the mediation of HA-Up (orf73) -HA-Down (orf73), the CRISPR-Cas9-lambda-red system inserts the relR-tat promoter into the phage orf73 to perform insertion and editing. Under the mediation of HA-Up (orf53) -HA-Down (orf53), the CRISPR-Cpf1-lambda-red system carries out replacement editing by relE-tat promoter. The experiment flow of the two editing methods is consistent Procedures are as follows:
Double Plate assay
Prepare for 1.5% agar plate containing particular antibiotics and 0.6% soft agar (10ml each tube) in advance. Notice that it’s the square petri dish marked with 6*6 checkerboard that should be used. Host bacteria should be cultured overnight in LB medium the night before experiment. On the day of the experiment, add Arabinose solution with final concentration of 0.1% to 2ml bacterial solution, which was then placed in a 37°C shaker to induce expression for 1 hour. In this hour, melt soft agar in 90°C water and keep it melted in 50°C water. Meanwhile, perform gradient dilution of phage with SM buffer (a kind of buffer where phage could exist). After that, take 0.1ml bacterial solution mentioned above, add Arabinose solution with final concentration of 10% and IPTG solution with final concentration of 1mM, pour one tube of melted soft agar in, shake it entirely and pour it on 1.5% agar plate. Note that the whole operation must be done swiftly, otherwise soft agar may get solidification in tube. When agar get solidification, drop 10UL diluted phage in the center of selected grids in the petri dish. After standing for 30min, invert petri dishes in 37°C incubator for 16~20 hours. Observe the plaque.
Then we need to screen for the successfully edited phage. Plaque PCR assay is used to achieve our goal. Procedures are as follows:
Plaque PCR assay
Prepare for enough 1.5ml EP tube and add 200ul SM buffer and 2ul chloroform to each tube. Pick up the plaque with a straw and transfer it to the solution mentioned above. Let set for an hour to fully lyse host cell and release the phage. Next, heat the EP tubes at 95°C in thermomixer for 10 minutes to fully lyse phages’ capsid protein. Then, centrifuge them at 12000rpm for 5 minutes. Phage genome is released in the supernatant. Amplify the DNA fragments meant to be inserted into the phage genome via PCR. PCR products are verified by DNA agar gel electrophoresis.
In order to further test the titer of the edited phages, it is necessary to purify the successfully edited phages. We purified phages as follows:
Phage Purification assay
Independent plaque was selected from an appropriate plate into a centrifuge tube containing 1ml SM buffer solution for gradient dilution. Add 0.6%LB soft AGAR (47°C) into the test tube containing 0.1ml host bacteria, mix well, pour into 1.5%LB AGAR plate, and place for 10min to solidify. Phage dilution drops were added to different areas of the plate from low concentration to high concentration. The plate was inverted at 37°C overnight. Plaque PCR assay mentioned above was used to select the phage with higher purity. Repeat until the phage is almost completely purified.
After the purified successfully-edited phages were obtained, it's necessary to prepare for phage progenies before they could be used for other experiments.
Preparation of phage progenies
0.1ml of the phage suspension was added to the test tube containing 0.1ml host bacteria, and incubated at 37°C for 15min. Then, 4ml preheated LB medium was added and shaken at 37°C at 180rpm until the host bacteria lysed. 100ul chloroform was added to promote the lysis of host bacteria. The bacteria were further shaken for 15min at 37°C and centrifuged at 4000g for 10min at 4°C to remove bacterial debris. The supernatant was recovered and 50ul chloroform was added and stored at 4°C.
Phage titer assay
Serially dilute phage progenies and add 0.1ml each diluted degree
Titer (PFU /mL) = plaque number * dilution ratio / 0.1
Proof of function
insertion editing
>Via double plate assay, we have got several plates with plenty of plaques:
Totally we have got 8 plates. In order to screen for the successfully edited phage, we decided to pick up plaques as much as possible. Eventually we picked up 18 plaques each plates, 144 plaques totally. Theoretical length of PCR product is 891bp. Actually, only band will appear in the gel map if the DNA fragment is successfully inserted in phage's genome. From gel map result,it's surprising to find that almost all the phages we picked up were edited successfully, which indicates that lambda-red system shows high recombination efficiency in the case of phage.
Then we performed phage purification assay and repeated it for two times. Three gel maps are shown below.
We used two pairs of primers to test the purity of phage: primer 5-F/045R were used to test whether successfully edited phage’s genome existed in the sample, while primer 5-F/ primer 5-R were used to test the purity of successfully edited phage. If there is almost no edited phage, only fragments with 1300bp could be found on the gel map. On the contrary, fragments with 1800bp could be found. From the three gel maps shown above we could find that with the process of phage purification, more and more fragments with 1800bp appear. On the third map, no fragments with 1300bp could be found, indicating that edited phages were successfully purified.
Next, phage titer assay was performed. We have got a series of plate with lots of plaques, but only the plate with 30~300 plaques will be used to calculate the phage titer. Finally, one plate, which dilution ratio equals to 10^7, was selected:
According to the formula: Titer (pfu /mL) = plaque number * dilution ratio / 0.1,
titer (pfu /mL) = 290 * 10^7 /0.1 = 2.9*10^10 pfu/mL
replacement editing
>Via double plate assay, we have got several plates with plenty of plaques:
In the same way, we performed plaque PCR assay to screen for the successfully edited phage:
According to our primer design, as long as there are bands, the presence of a successfully edited phage genome in the sample can be demonstrated.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2756
Illegal PstI site found at 2251
Illegal PstI site found at 2498 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2756
Illegal PstI site found at 2251
Illegal PstI site found at 2498 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2756
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2756
Illegal PstI site found at 2251
Illegal PstI site found at 2498 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2756
Illegal PstI site found at 2251
Illegal PstI site found at 2498
Illegal AgeI site found at 2678 - 1000COMPATIBLE WITH RFC[1000]
Reference
[1]Bagdasarian, M. M., Amann, E., Lurz, R. & Rückert, B. Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-host-range, controlled-expression vectors. Gene 26, 273-282 (1984).
[2]de Boer, H. A., Comstock, L. J. & Vasser, M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A 80, 21-25, doi:10.1073/pnas.80.1.21 (1983).
[3]Jeong, J., Cho, N., Jung, D. & Bang, D. Genome-scale genetic engineering in Escherichia coli. Biotechnol Adv 31, 804-810, doi:10.1016/j.biotechadv.2013.04.003 (2013).
[4]Court R, Cook N, Saikrishnan K, Wigley D (2007). The crystal structure of lambda-Gam protein suggests a model for RecBCD inhibition. JOURNAL OF MOLECULAR BIOLOGY 371: 25-33.
[5]Murphy KC (1998). Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. JOURNAL OF BACTERIOLOGY 180: 2063-2071.
[6]Murphy KC (2016). lambda Recombination and Recombineering. EcoSal Plus 7.
[7]Passy SI, Yu XN, Li ZF, Radding CM, Egelman EH (1999). Rings and filaments of beta protein from bacteriophage lambda suggest a superfamily of recombination proteins. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 96: 4279-4284.
[8]Poteete AR (2001). What makes the bacteriophage lambda Red system useful for genetic engineering: molecular mechanism and biological function. FEMS MICROBIOLOGY LETTERS 201: 9-14.
[9]Zhang J, McCabe KA, Bell CE (2011). Crystal structures of lambda exonuclease in complex with DNA suggest an electrostatic ratchet mechanism for processivity. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 108: 11872-11877.
Luk, K. C. & Szybalski, W. Characterization of the cloned terminators tR1, tL3 and tI and the nut R antitermination site of coliphage [10]lambda. Gene 20, 127-134, doi:10.1016/0378-1119(82)90030-0 (1982).
[11]Luk, K. C. & Szybalski, W. Transcription termination: sequence and function of the rho-independent tL3 terminator in the major leftward operon of bacteriophage lambda. Gene 17, 247-258, doi:10.1016/0378-1119(82)90140-8 (1982).
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