Team:Hong Kong HKU/results
RESULTS - WET LAB
The Design
After we identified our research goal, which is to achieve a control over the ratiometric distribution
of two or more phenotypes of bacteria within a culture, we made use of the inversion property of Cre-lox
and avoided any deletion event in order to preserve all of the possible phenotypes in the gene pool.
We designed the lox-reporter constructs and cre mutant constructs with Snapgene and Geneious Prime.
Figure 1 illustrates the general structure of the lox-reporter construct. For easier observation of the
flipping events, we used fluorescent reporter genes such as green fluorescent protein (GFP) and red
fluorescent protein (RFP) for colorimetric analysis. To minimize the metabolic burden given by the
lox-reporter construct, we placed an inducible promoter upstream of the first lox sites.
Figure 1: Design of lox-reporter construct. In the expression cassette, the orthogonal lox sites sandwich two distinctive sets of reporter gene cassettes. GFP and RFP genes are in different directions, so only one gene will be expressed at a time. For irreversible recombination events, GFP (forward) will be expressed in the absence of Cre whereas RFP will instead be expressed after being flipped by Cre.
On the other hand, we also created a variety of cre mutants by site-specific mutagenesis, with reference to Dr Eroshenko's paper.1In the pursuit of improving the specificity of the Cre-based recombination, we used various Cre mutants in our project, instead of the wild types. The cre recombinase mutants are designed to have one amino acid difference as compared to the wildtype. The change in amino acid sequence may slightly alter the cre conformation, in particular the active sites. The cre mutants used include Cre-R32V, Cre-R32M, 303GVSdup and Cre-Y324F.
With Pymol, the wild-type Cre recombinase and all the mutated versions (specific residues highlighted) could be visualized. Figure 2 shows the wild-type Cre recombinases which form a dimer around a strand of DNA. Figure 3 shows all the Cre mutants used in our experiment (R32M, R32V, Y324F and 303GVSdup). According to the paper by Dr Eroshenko, three of the mutants (R32M, R32V and 303GVSdup) shows higher specificity as the mutation destabilizes the linkage between the 2 dimers. As the authors suggest, instead of disturbing the DNA-protein interaction, dimer interaction is a key to Cre recombinase specificity. We did a meeting with Dr Eroshenko discussing about his paper and our project Link Here
Figure 2: 3D structure of Cre recombinase dimer interacting with a strand of DNA. Pymol was used to visualize the crystal structure of specific protein with PDB files available in online databases.
Figure 3: 3D structure of Cre recombinase mutants. A-B: Cre R32M mutant; C: 303GVSdup mutant (mutated segment hightlighted in white circle); D-E: Cre R32V mutant; F: Y324F mutant (mutated residues hightlighted in white circle). As the structure and residues change suggest, destabilizing dimer interaction could enhance specificity.
In addition to the mutations created on Cre recombinases, variation is also added to the ribosome binding site (RBS). A stronger RBS refers to a more efficient translation of the mRNA and therefore high expression level of Cre. In this project, strong and weak RBS are incorporated to the Cre-mutant construct for the determination of the metabolic burden and recombination efficiency.
Figure 4: Design of Cre-mutant constructs. The expression of Cre recombinase is controlled by the level of arabinose. Mutation would also be introduced to the Cre gene for improving the specificity of cre binding and in turns increasing recombination efficiency.
The experiments
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Amplification of parts
The biobricks involved in this project are mainly from the iGEM distribution kit and IDT oligo. After we received the distribution kits, we first amplified the essential biobricks such as the lox sites and Cre-encoding sequences, as well as the backbones with different resistance and copy numbers. (state more details here first mb, like explain or show the Snapgene file for psb1c3)
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Site-directed mutagenesis on Cre recombinase
We used 4 sets of specific primers that would introduce desirable mutation to wild-type Cre during polymerase chain reaction (PCR). The mutations include point mutations (R32M, R32V and Y324F) and duplication (303GVSdup).
Sanger sequencing in both forward and reverse directions showed that we successfully created four cre mutants which were then incorporated into the corresponding backbones containing ribosome binding sites (RBS) and arabinose-inducible promoter.The alignment result showed that all of the mutations had been successfully and accurately introduced to the Cre gene individually by site-directed mutagenesis.
Figure 5: Multiple alignment between WT Cre and Cre mutants. A three-nucleotide change was introduced to the Cre gene which will encode for different amino acids at position 32 of the protein sequence.
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Lox-reporter construct and Cre construct assembly
Each biobrick contains a prefix (containing EcoRI and XbaI restriction sites) and suffix (containing SpeI and PstI restriction sites) and has been arranged in a standard psB1C3 plasmid. We extracted the biobricks with appropriate restriction enzymes and assembled them through DNA ligation.
We assembled the lox-reporter constructs and the cre mutant constructs by stepwise molecular cloning -- adding the ribosome binding site (RBS), the promoters and the reporter genes and the terminators, bit-by-bit into the psB1C3 backbone (Figure 1 and Figure 2). The recombinant plasmid was then transformed into the DH5a E. Coli strain and the selected colonies went through colony PCR and was amplified by bacterial culture. The transformants were verified by restriction enzyme digestion followed by agarose gel electrophoresis. Only when the restriction fragments match the expected size of the vector and that of the inserted gene could the cloning be said to be successful (figure 7). The presence of two fragments (insert & backbones) in the lane indicates the successful digestion of the construct. The respective size of the fragments further implies the presence of all essential components in the construct. All of the cre mutants were correctly cloned with all of the essential components. However, only loxP and lox2272 among all of the lox-reporter constructs showed the correct fragment sizes.
Figure 6: Schematic of the stepwise cloning
Figure 7: Gel electrophoresis for digestion products of lox-reporter constructs (Left) and cre-mutant constructs (Right).
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Switching backbones
We created lox-reporter constructs and Cre-mutant constructs in psB1C3 backbone which is a high copy number plasmid containing chloramphenicol resistance. As double antibiotics resistance is required for co-transformation at the later stage for testing the ratiometric control of lox-cre recombination, we needed to switch the cre-mutant and lox-reporter to different backbones. We switched the lox-reporter construct to pSB1A3 (high copy number, ampicillin resistance). For Cre mutants, we selected plasmids with chloramphenicol resistance and assigned the copy number according to the strength of the RBS -- strong RBS with low copy number plasmid (i.e. psB4C5) and weak RBS with high copy number plasmid (i.e. psB1C3). This is to even out the final expression level between Cre-mutant constructs with strong RBS and those with weak RBS to avoid metabolic overload to the competent cells.
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Co-transformation and first experiment
For the validation of our lox-Cre system, we intend to first co-transform the lox-reporter and cre mutants into the competent cells for lox-cre recombination to occur, and then induce the Cre expression with different concentration of arabinose. The validation ends by re-transforming the lox-reporter to visualize the colour of the colonies. The ratio of Green colonies: Red colonies would imply the Cre-lox recombination efficiency. (However, due to the COVID-19 outbreak, the wetlab progress remains stagnant and real experiment could not be done properly.)
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In vitro Wild-Type Cre shuffling
Figure 8: Showing the gel of reporter construct with restriction digestion checking of (a) Deletion events (EcoRI, PstI E+P), and (b) Flipping Events (EcoRI, BsrGI E+B). Also displaying the copy number variation for the time to reach complete equilibrium with variation of substrate DNA amount.
The Cre shuffling was conducted with 1 unit of Wild Type Cre in 37C for 30 minutes, with 125ng of template as 1X substrate. With a wild type Cre, there is a miscellaneous band at ~750bp, which indicates the loxP sites interacting and recombining with a plasmid backbone sequence. If it is a deletion due to 2 loxP sites, the expected size would be 260bp with EcoI and PstI digestion, which is not present in the gel. It indicates the deletion is likely to be with an off-target site. This re-asserts the importance of the use of mutant versions of Cre in our system, as discussed in the meeting with Dr Eroshenko.
With Image J, we could calculate the amount of flipped construct versus initial construct in 1X substrate.Band Intensity Length Intensity/Length Percentage of Inversion E+B Initial Backbone 33727 3662 9.21 50.547% E+B Inverted Backbone 19397 2476 7.83 / E+B Inverted Fragment 19327 1758 10.99 / Table 1: Ratio of unflipped construct to flipped construct Therefore, we could see it reach equilibrium state within 30 minutes.
Our reporter construct used in the pilot testing would be a high-copy number plasmid (100-300 copies) which directly extends the time to reach equilibrium, as illustrated by this experiment. With a normal 1 unit Cre with 0.125 ug construct with 2 loxP sites, the shuffling went to equilibrium state. However, the others with 10X or 100X copy number, there is no sign of flipped construct if equal DNA amount is loaded to gel, indicating much slower progress for all to equilibrium position of 50%.
Hence, if we were to use the shuffling system with a single copy gene introduced with a lambda red system, it will only need a hundredth of the time needed to reach equilibrium. Therefore, it also provides good dry lab data to simulate rate and probability of flipping with a high-copy number plasmid model, after in vivo testing. -
In vivo Wild-Type Cre shuffling
Figure 9: Showing the restriction digestion results of plasmids extracted after arabinose induction. EP indicates EcoRI + PstI digestion, and EB indicates EcoRI + BsrGI digestion. WT = Cre wild type. R32V, R32M, 303dup = Cre mutants with high specificity. Y324F = non-active Cre mutants.
The reporter construct and the Cre constructs (high copy, weak RBS) was co-transformed into NEB stable E. Coli cell. The culture was grown in 0.2% arabinose with cells extracted at 30 minutes, 2.5 hour, 4 hour, 16 hour, 24 hour and 48 hour, in presence of chloramphenicol and carbenicillin. Cells extracted were spin down, washed twice and incubated with medium with carbenicillin in LB, growing until the 24 hour mark. Plasmids were extracted with a mini-prep kit and analysed with restriction digestion.
From the result, we could see mutant versions have less genome targeting activity in a same time-frame. From the left, we could see the deletion band (Red 1), the absence of the deletion band indicates the complete loss of the biobrick in the vector, since it represents the EcoRI + PstI digestion. It indicates the integration of the whole reporter plasmid into the bacterial genome. From the WT EB cut, we could also see the absence of the reporter plasmid in the Red 2. It shows the instability of maintaining lox construct in genome level or plasmid with wild type Cre, due to the cross-reactivity with genome off-target sites.
Cre-mutants show a much lower extent of genome off-target effect. However, due to possible over-expression of Cre which leads to excision instead of inversion, we could not observe correct inversion patterns (Green box), but only excision patterns (Red 1) in EP cut. The non-cutting control shows correct banding of the initial position cut (Blue box).
This shows we will need to adjust and lower the Cre expression level, since from the original Cre-mutant paper, the inversion could be maintained without detectable excision pattern [1]. Studies also show with too much Cre inside a system, deletion would occur instead of inversions by losing the directionality of lox sites, which leads to our observation[2]. Even with a weak RBS, the cre expression is too high, therefore the approach would be to decrease the arabinose concentration to further tune and reduce cre mutant expression.
This also gives an initiative to investigate into other recombinase systems which does not involve deletion, including Hin, Gin, Fin orthogonal systems, which could use the same model and design to achieve the same goal.To further optimize the Cre expression level, we induced the Cre with different arabinose to evaluate the activity and stability of the cre mutants.
Figure 6. Optimisation of Cre mutant expression level. Showing restriction digestion check of Cre (co-transformed with reporter construct) activity in vivo. The bands expected were produced by reporter construct and are not present in Cre constructs.
On the left, we could observe inversion bands in R32V mutant at 0.01% but not 0.1% or 0.001% of arabinose expression. Too much Cre inhibits the inversion rate via blocking of lox sites, while too few Cre decreases the rate of reaction. We could also see the wild type Cre has no pattern in all arabinose concentration, either inverted or initial position, indicating loss of genetic information by wild type.
Band Intensity Corresponding bp Normalized intensity Corresponding flipping rate (flipped) 27610 2480 11.13 55% 23960 1750 13.69 Table 7. R32V 0.01% arabinose flipping percentage measured via imageJ After we determined the optimal arabinose concentration, we ran all mutants with wild type for digestion (Same batch and same samples).
Figure 8. Showing the digestion pattern (0.01% arabinose) of all mutants in vivo extending from figure 7.
Orange shows the wild type, which shows that the reporter construct has disappeared or integrated into the genome via nonspecific interactions (Orange). Meanwhile, the mutants retain either initial or deleted conformation. However, it is of our surprise we were able to observe inversion patterns but restriction digestion with EcoRI and PstI shows a deleted position compared to Y324F control (Green). On the right, we could observe inversion position bands (Blue), and the initial position bands (Red). While the last figure 7 and this figure 8 uses the same sample and restriction digestion, and we are able to observe both band patterns in mutants in figure 7, the DNA bands may have overlapped for the mutants in figure 8‘s gel.
From these experiments, we could conclude several things. Cre mutants have overwhelming advantages compared to Cre wild type in terms of specificity and accuracy of inversion, as we see Cre wild type has a loss of reporter construct in all in vivo experiments. Meanwhile, we could observe inversion and initial patterns in Cre mutants. However, there are still minor deletion events shown and we would improve our project by implementing our proposed solutions.
The future
There are few things that we want to accomplish in coming year:
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Prove that our system works by completing the co-transformation and analyse the green: red colonies.
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Get into a more complex design of the lox-Cre recombinase.
Figure 10: A more complex design of our lox-cre system.
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crRNA cas13 designs to test and eliminate intermediate zones.
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Use of recombinases Bxb1 excisionase and incisionase instead of the Cre for 2 phenotypes with higher accuracy and control, with complementary codon optimisation and import signal for eukaryotic systems.
References:
- Eroshenko N, Church GM. Mutants of Cre recombinase with improved accuracy. Nature communications. 2013 Sep 23;4(1):1-0.
- Campo N, Daveran-Mingot ML, Leenhouts K, Ritzenthaler P, Le Bourgeois P. Cre-loxP recombination system for large genome rearrangements in Lactococcus lactis. Applied and Environmental Microbiology. 2002 May 1;68(5):2359-67.