Nov 18, 2020
Intein-assisted Bisection Mapping (IBM)
- 1University of Edinburgh
Protocol Citation: Trevor YH Ho, Baojun Wang 2020. Intein-assisted Bisection Mapping (IBM). protocols.io https://dx.doi.org/10.17504/protocols.io.bpqdmms6
Manuscript citation:
Ho, T.Y.H. et al. A systematic approach to inserting split inteins for Boolean logic gate engineering and basal activity reduction. Nature Communications 12, 2200 (2021).
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 14, 2020
Last Modified: November 18, 2020
Protocol Integer ID: 44517
Keywords: split protein, split intein, bisection mapping, logic gates
Disclaimer
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Abstract
Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augmented a mini-Mu transposon-based screening approach and devised the intein-assisted bisection mapping (IBM) method. IBM robustly revealed clusters of split sites on four proteins, converting them into AND or NAND logic gates. In this protocol we provide details in executing the IBM method.
Guidelines
It would be best to read the manuscript "Intein-assisted bisection mapping systematically splits proteins for Boolean logic and inducibility engineering" prior to engaging this protocol.
An overview of protocol is given in Introduction. Please peruse before proceeding to the rest of the protocol. Please also refer to our manuscript for the literature on which this method was built upon.
Materials
Most materials used in this protocol can be obtained from common commercial vendors, except the MuA transposase.
While theMuA TransposaseThermo FisherCatalog #F750 is available, our MuA transposase was purified and supplied by Domus Biotechnologies (Turku, Finland).
With the transposase comes with:
- MuA dilution buffer
- 2x MIX
The following solution needs to be prepared by need to be prepared by users beforehand
- 2.5 M NaCl + 0.25 M MgCl2 1μl
- 1.25% Triton X-100
Wherever necessary, users might need to prepare their own electrocompetent cells.
Other reagents and materials used in this protocol:
Kits and consumables
Gene Pulser/MicroPulser Electroporation Cuvettes 0.1 cm gapBio-rad LaboratoriesCatalog #1652089
NEB 10-beta Electrocompetent E.coli - 6x0.1 mlNew England BiolabsCatalog #C3020K
NEB 10-beta/Stable Outgrowth Medium - 4x25 mlNew England BiolabsCatalog #B9035S
QIAprep Spin Miniprep Kit PrintQiagenCatalog #27104
ZymoPURE™ II Plasmid Midiprep KitZymo ResearchCatalog #D4201
Monarch PCR and DNA Cleanup Kit - 250 prepsNew England BiolabsCatalog #T1030L
Aluminium SealStarLabCatalog #E2796-9792
Inducers:
L-( )-ArabinoseSigma AldrichCatalog #A3256
24-DiacetylphloroglucinolCambridge BioscienceCatalog #16345
Enzymes:
BsaI-HFv2New England BiolabsCatalog #
R3733L
FastAP Thermosensitive Alkaline Phosphatase (1 U/µL)Thermo FisherCatalog #EF0651
BbsI-HF - 1,500 unitsNew England BiolabsCatalog #R3539L
BglII - 10,000 unitsNew England BiolabsCatalog #R0144L
SapI - 1,250 unitsNew England BiolabsCatalog #R0569L
T4 DNA LigaseNew England BiolabsCatalog #M0202
Taq DNA Polymerase with ThermoPol® BufferNew England BiolabsCatalog #M0267L
Equipment (necessary):
Equipment
MicroPulser Electroporator
NAME
Electroporator
TYPE
Bio-Rad laboratories
BRAND
1652100
SKU
Equipment (optional):
Equipment
Eppendorf™ Concentrator Plus with Pump and GB Plug
NAME
Concentrator
TYPE
Eppendorf
BRAND
5305000568
SKU
Equipment
OT-2
NAME
Liquid handler
TYPE
Opentrons
BRAND
OT-2
SKU
Protocol materials
FastAP Thermosensitive Alkaline Phosphatase (1 U/µL)Thermo FisherCatalog #EF0651
L-( )-ArabinoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #A3256
MuA TransposaseThermo FisherCatalog #F750
Gene Pulser/MicroPulser Electroporation Cuvettes 0.1 cm gapBio-Rad LaboratoriesCatalog #1652089
BsaI-HFv2New England BiolabsCatalog #
R3733L
ZymoPURE™ II Plasmid Midiprep KitZymo ResearchCatalog #D4201
Monarch PCR and DNA Cleanup Kit - 250 prepsNew England BiolabsCatalog #T1030L
24-DiacetylphloroglucinolCambridge BioscienceCatalog #16345
SapI - 1,250 unitsNew England BiolabsCatalog #R0569L
Taq DNA Polymerase with ThermoPol® BufferNew England BiolabsCatalog #M0267L
NEB 10-beta/Stable Outgrowth Medium - 4x25 mlNew England BiolabsCatalog #B9035S
Aluminium SealStarLabCatalog #E2796-9792
BglII - 10,000 unitsNew England BiolabsCatalog #R0144L
NEB 10-beta Electrocompetent E.coli - 6x0.1 mlNew England BiolabsCatalog #C3020K
BbsI-HF - 1,500 unitsNew England BiolabsCatalog #R3539L
T4 DNA LigaseNew England BiolabsCatalog #M0202
QIAprep Spin Miniprep Kit PrintQiagenCatalog #27104
FastAP Thermosensitive Alkaline Phosphatase (1 U/µL)Thermo FisherCatalog #EF0651
BsaI-HFv2New England BiolabsCatalog #
R3733L
BglII - 10,000 unitsNew England BiolabsCatalog #R0144L
BsaI-HFv2New England BiolabsCatalog #
R3733L
NEB 10-beta Electrocompetent E.coli - 6x0.1 mlNew England BiolabsCatalog #C3020K
Gene Pulser/MicroPulser Electroporation Cuvettes 0.1 cm gapBio-Rad LaboratoriesCatalog #1652089
NEB 10-beta/Stable Outgrowth Medium - 4x25 mlNew England BiolabsCatalog #B9035S
QIAprep Spin Miniprep Kit PrintQiagenCatalog #27104
ZymoPURE™ II Plasmid Midiprep KitZymo ResearchCatalog #D4201
Monarch PCR and DNA Cleanup Kit - 250 prepsNew England BiolabsCatalog #T1030L
BsaI-HFv2New England BiolabsCatalog #
R3733L
T4 DNA LigaseNew England BiolabsCatalog #M0202
T4 DNA LigaseNew England BiolabsCatalog #M0202
BbsI-HF - 1,500 unitsNew England BiolabsCatalog #R3539L
SapI - 1,250 unitsNew England BiolabsCatalog #R0569L
BbsI-HF - 1,500 unitsNew England BiolabsCatalog #R3539L
SapI - 1,250 unitsNew England BiolabsCatalog #R0569L
Gene Pulser/MicroPulser Electroporation Cuvettes 0.1 cm gapBio-Rad LaboratoriesCatalog #1652089
L-( )-ArabinoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #A3256
24-DiacetylphloroglucinolCambridge BioscienceCatalog #16345
Aluminium SealStarLabCatalog #E2796-9792
Taq DNA Polymerase with ThermoPol® BufferNew England BiolabsCatalog #M0267L
Safety warnings
No particular hazard in executing this protocol.
Observe common biosafety precautions.
Before start
Before you start, you will need to perform some molecular cloning on your gene of interest. You need to separate most of the middle part of the coding DNA sequence (termed slightly trimmed CDS in this protocol) and subclone it onto a staging vector.
Plasmid construction prior to IBM execution.
This ensures that all transposition events happen within the CDS of your choice. Please also note how the transposition window was defined. In this protocol, most of the steps will be illustrated on an example of conducting intein-assisted bisection mapping on the gene encoding TEM-1 β-lactamase, where the protein will be split by the gp41-intein such that both the N-lobe (our way of calling the N-terminal split protein fragment) and the C-lobe need to be expressed to gain resistance to ampicillin.
Introduction
Introduction
Summary workflow of IBM. This overview describes the split of β-lactamase by the gp41-1 intein.
IBM starts with a transposition reaction: a staging vector carries a 5’ and 3’ trimmed gene of interest (GOI) with the internal BsaI, BbsI and SapI sites removed. The staging plasmid is mixed in vitro with the MuA transposase and the mini-Mu transposon, which generates the initial insertion library. After in vivo amplification (transformation into E. coli and overnight growth), the insertion library is cut by BsaI and resolved on a DNA agarose gel. The band with the correct size corresponding to GOI with the insertion is purified and ligated into a linearized, protein expression vector with BsaI-generated overhangs. This produces the Open Reading Frame (ORF) insertion library. The ORF insertion library is again amplified in vivo and then an aliquot is mixed with a DNA fragment carrying the split gp41-1 intein intein in a Golden Gate reaction, using the restriction enzymes BbsI and SapI. The DNA fragment contains a different selection marker (cat) to facilitate selection of the library with the transposon replaced. In the end, the final library is screened for individual strains that showed proper reconstitution of protein function, i.e. resistance against ampicillin upon expression of both the N- and C-lobes (AND logic gate behavior). Those strains were then subjected to Sanger sequencing the C-terminal joint to map the split sites. Identical split sites were then deduplicated and consolidated with activity data to generate the intein-bisection map.
This protocol describes the main procedures of transposition, size selection, substitution, and mapping. Molecular cloning to generate the starting DNA constructs are not covered. However, this protocol will start with describing the necessary treatment on those constructs once they are available.
The most important objective in this protocol is to obtain a final library with large enough "library complexity, or diversity", in other words, ensuring that during construction and the final phase, the libraries contain enough random events so one can be confident that most possible splits are covered, and that a function split site for intein can be screened out.
Kits
Kits
We found good quality of DNA from each step is essential for the success of the protocol.
We use the following kits:
- QIAprep Spin Miniprep Kit PrintQiagenCatalog #27104 for miniprep
- ZymoPURE™ II Plasmid Midiprep KitZymo ResearchCatalog #D4201 for midiprep
- Monarch PCR and DNA Cleanup Kit - 250 prepsNew England BiolabsCatalog #T1030L for gel and all other DNA purifications and concentrating steps.
Preparation of DNA materials
Preparation of DNA materials
2h
2h
Prepare linearized protein expression vector
Grow and harvest 2 * 10 mL bacterial culture of the protein expression vector.
Perform a mini-prep the next day.
Pool the extracted DNA into one tube.
Digest 5 µg plasmid of protein expression vector from miniprep usingBsaI-HFv2New England BiolabsCatalog #
R3733L .
Simultaneously treat with FastAP Thermosensitive Alkaline Phosphatase (1 U/µL)Thermo FisherCatalog #EF0651 .
| Number of reactions | 1 | 5 | |
| 10X Cutsmart Buffer | 2 | 10 | |
| BsaI-HFv2 (20 U/μL) | 1 | 5 | |
| FastAP (1 U/μL) | 1 | 5 | |
| DNA (concentration: 89 ng/μL) | 11.2 | 56 | |
| water | 4.8 | 24 |
Example digestion recipe for linearizing the protein expression vector. All volumes in tables are in μL.
Run reaction at 37 °C for at least 02:00:00 .
2h
Resolved digested DNA on 0.5 - 1% agarose gel, depending on the size of the vector. Load 1 µg digested DNA per lane.
Excise the band corresponding to the linearized vector.
Purify the DNA.
Load at most 2 gel slices (1 slice per lane on gel) per column.
Elute DNA from each column using 10 µL nuclease free water .
Pool eluted DNA together and quantify the concentration.
Prepare the linearized mini-Mu transposon
Grow and harvest 50 mL bacterial culture for the plasmid carrying the MuA-transposon (plasmid ID = IBMc090).
Perform a midi-prep the next day.
Digest 20 µg mini-Mu containing plasmid from miniprep usingBglII - 10,000 unitsNew England BiolabsCatalog #R0144Laand BsaI-HFv2New England BiolabsCatalog #
R3733L .
| 1X | 20 | ||
| 10X Cutsmart Buffer | 2 | 40 | |
| DNA (1000 ng/μL) | 1 | 20 | |
| BglII (10 U/μL) | 1 | 20 | |
| BsaI-HFv2 (20 U/μL) | 1 | 20 | |
| water | 15 | 300 |
Example digestion recipe for linearizing the mini-Mu transposon. All volumes in tables are in μL.
Run reaction at 37 °C , for at least 02:00:00 , orOvernight if time is permitted.
2h
Resolved digested DNA on 1% agarose gel. Load 1 µg digested DNA per lane.
Run the gel at slow speed, e.g. 75 V for 60 - 90 mins helps to increase band sharpness.
Expected result
Expected result of digested IBMc090 resolved on agarose gel.
Excise the band with size of 1667 bp, which corresponds to the linear transposon.
Purify the DNA.
Load at most 2 gel slices (1 slice per lane on gel) per column.
Elute DNA from each column using 10 µL nuclease free water
Pool eluted DNA together and quantify the concentration.
Expected result
It is important to obtain > 80 ng/μL of purified linear transposon for downstream processes.
If your purification efficiency is too low, try using a
Equipment
Eppendorf™ Concentrator Plus with Pump and GB Plug
NAME
Concentrator
TYPE
Eppendorf
BRAND
5305000568
SKU
to concentrate the DNA.
Prepare the staging plasmid
Grow and harvest 10 mL bacterial culture for the staging plasmid.
Perform a mini-prep the next day.
Prepare the linear substitution insert DNA
Grow and harvest 50 mL bacterial culture for the plasmid carrying the substitution insert.
Perform a midi-prep using the next day.
Digest 10 µg substitution insert containing plasmid from midiprep usingBbsI-HF - 1,500 unitsNew England BiolabsCatalog #R3539L and SapI - 1,250 unitsNew England BiolabsCatalog #R0569L
| NEB buffers and enzymes | 1 | 10 | |
| 10X CS Buffer | 2 | 20 | |
| DNA (819 ng/μL) | 1.2 | 12 | |
| BbsI-HF (20U/μL) | 1 | 10 | |
| SapI (10U/μL) | 1 | 10 | |
| ddH2O | 14.8 | 148 |
Example recipe to release substitution insert. All volumes in tables are in μL.
Note: DO NOT try to be thrift with SapI. It will result in incomplete digestion.
Run reaction at 37 °C , for at least 02:00:00 , orOvernight if time is permitted.
2h
Resolved digested DNA on 1% agarose gel. Load 1 µg digested DNA per lane.
Expected result
Excise the band with size of 2591 bp, which corresponds to the substitution insert.
Purify the DNA.
Load at most 2 gel slices (1 slice per lane on gel) per column.
Elute DNA from each column using 10 µL nuclease free water
Pool eluted DNA together and quantify the concentration.
Transposition using an engineered mini-Mu transposon
Transposition using an engineered mini-Mu transposon
6h 50m
6h 50m
Prepare MuA transposase
The MuA transposase should be stocked at -80 °C at high concentrations to maximize shelf-life.
Dilute the concentrated MuA transposase to 220 ng/μL right before setting up the transposition reaction.
Using leftover MuA transposase from previous experiments, even if kept at -20 °C , could lead to a much reduced transposition efficiency.
Set up 6 * 25 µL transposition reaction .
| Number of reactions | 1 | 6 | |
| 2X Mix | 12.5 | 75 | |
| 2.5 M NaCl + 0.25 M MgCl2 | 1 | 6 | |
| 1.25% Triton X-100 | 1 | 6 | |
| MuA Transposase (220 ng/μl) | 3 | 18 | |
| Transposon (69.6 ng/μl) | 4.3 | 25.8 | |
| Vector (158 ng/μl) | 1.3 | 7.8 | |
| H2O | 1.9 | 11.4 |
Example recipe for transposition reaction. All volumes in tables are in μL.
Aliquot all mixtures into 2 * 75 µL reactions in 200 μl PCR tubes.
Run reactions on a thermocycler for
1. 30 °C for 06:00:00
2. 80 °C for 00:10:00
6h 10m
Pooled the reaction mixture together and purify the DNA library.
Use a single column and elute all DNA in10 µL nuclease free water .
It is not necessary to quantify the DNA concentration.
Electroporation of DNA library into E. coli
Electroporation of DNA library into E. coli
Electroporate the DNA library into NEB 10-beta Electrocompetent E.coli - 6x0.1 mlNew England BiolabsCatalog #C3020K .
Aliquot 4 * 1 mL ofNEB 10-beta/Stable Outgrowth Medium - 4x25 mlNew England BiolabsCatalog #B9035S (come with the electrocompetent cells) into microcentrifuge tubes or 30 mL universal tubes / test tubes and warm up the medium in tubes at 37 °C for at least00:30:00 .
30m
Thaw and then mix 100 µL electrocompetent cells with the eluted DNA library in a pre-chilled microcentrifuge tube. Place the tube on ice for 00:05:00
5m
Aliquot 27.5 µL DNA-competent cell mixture into a Gene Pulser/MicroPulser Electroporation Cuvettes 0.1 cm gapBio-Rad LaboratoriesCatalog #1652089
Ensure that the mixture is loaded in the gap. Place the cuvette on ice for 00:05:00
5m
Take out the warmed up Stable Outgrowth Medium.
Wipe down any traces of water on the cuvettes. Carry out electroporation using
Equipment
MicroPulser Electroporator
NAME
Electroporator
TYPE
Bio-Rad laboratories
BRAND
1652100
SKU
Use protocol EC1. Between each cuvette, jot down the time constants for electroporation event. The value should be between 4 to 5.5 ms. Even if the value is out-of-range, keep going.
Immediate after electroporation, add 972.5 µL Stable Outgrowth Medium into the electroporation cuvette. Gently suspend cells by mixing up and down for 1-2 times. Transfer all suspended cells in medium into the microcentrifuge or universal tube where the medium was warmed. For samples of the same library, pool the suspended cells together.
It may be helpful to do this aseptically, but this is not strictly required. Speed is more important.
After finishing the electroporation steps for all samples, incubate the tubes at 160 rpm, 37°C, 01:00:00 for recovery.
Retrieve the recovered cells.
For each library, there should be ~ 4 mL of recovered cells. Take out 20 µL recovered cells and dilute it into 180 µL 0.85% saline . Serially dilute it again 10-fold for 5 times. Label the tubes from A (lowest dilution) to E (highest dilution).
Spread 100 µL diluted cells onto 90 mm LB agar + KAN + CHL plates labelled A to E. This is to quantify the "library complexity".
For the rest of the recovered cells, spread onto 4 * 145 mm LB agar + KAN + CHL plates, each using ~ 1 mL recovered cells . Allow the plates to dry before inverting them upside down.
Incubate all plates overnight at 37 °C
Retrieve the plates and determine library complexity.
For each library, tally the colony counts from plates A - E and create a table like below:
Expected result
| Plate | # of colonies | dilution factor | Initial number of transformants | |
| A | TMTC | 400 | - | |
| B | TMTC | 4000 | - | |
| C | TMTC | 40000 | - | |
| D | 48 | 400000 | 19200000 | |
| E | 4 | 4000000 | 16000000 |
Example record table for calculating library complexity. TMTC = too many to count
For n bp there is n-1 possibilities of insertion, and the transposon can insert in both forward and reverse orientations.
Total number of colonies (aka library complexity) was 1.92 * 107.
Size of staging plasmid is 2859 bp.
Therefore, library fold coverage was 1.92 * 107/ ((2859 - 1) * 2) = 3359 fold
If the library fold coverage is > 100. Proceed. Otherwise, discard the library.
Cell scraping and midiprep
Cell scraping and midiprep
Scrap down all cells from the 4 * 145 mm plates using a L-shape spreader and 0.85% saline. For each plate, add 20 mL 0.85% saline to each plate, scrape down cells and transfer cells in saline to a 50 mL centrifuge tube. Repeat this once.
Centrifuge all tubes at 5000 rpm, Room temperature, 00:10:00 , remove most of the supernatant until the volume is < 10 mL , then resuspend the cell pellets completely. Pool all resuspended cells into one of the four tubes. Repeat centrifugation with a balance.
Remove supernatant again until total volume is about 5 mL . At this the suspension is very dense and probably a bit viscous. Take out 1 mL resuspended cells and save it as a glycerol stock.
The glycerol stock can be re-propagated and will save huge amount of time if you need to reuse the library.
Repeat the centrifuge again and this time, discard all supernatant. Perform a midiprep to extract the initial insertion library.
Size selection on initial insertion library to generate ORF insertion library
Size selection on initial insertion library to generate ORF insertion library
2h
2h
Digest 10 µg extracted, initial insertion library using BsaI-HFv2New England BiolabsCatalog #
R3733L
| Number of reactions | 1 | 10 | |
| 10X CS Buffer | 2 | 20 | |
| BsaI-HFv2 (20U/μL) | 1 | 10 | |
| DNA (769 ng/μL) | 1.3 | 13 | |
| water | 15.7 | 157 |
Example digestion recipe for size selection. All volumes in tables are in μL.
Run reaction at 37 °C , for at least 06:00:00 , orOvernight if time is permitted.
6h
Resolved digested DNA on 0.5 - 1% agarose gel. The gel percentage will depend on the expected sizes.
Load 1 µg digested DNA per lane.
Critical: Run the gel at slow speed, e.g. 75 V for 90 mins. Resolution between bands is extremely important here, because the band corresponding to the trimmed CDS inserted with the transposon could be faint and close to other bands.
Excise the band which corresponds to the trimmed CDS inserted with the linear transposon.
Example in calculating the theoretical size of the trimmed CDS inserted with the transposon
- The staging plasmid for β-lactamse is 2859 bp.
- When digested by BsaI, it yields 771 bp (trimmed CDS) and 2088 bp (backbone).
- Therefore, after transposition:, each receives + 1667 + 5 bp
- Expected sizes of digested library are therefore 771, 2088, 2443, 3760 bp
- The 2443 bp band is the band containing the trimmed CDS inserted with transposon
Expected result
Example size selection step during IBM on β-lactamse, pre-excision. Notice how faint the desired band could be.
Example size selection step during IBM on β-lactamse, post-excision. It might not be always possible to exclude all other fragments while recover everything single bit of the desired band, but it is ok to proceed.
Purify the DNA.
Load at most 2 gel slices (1 slice per lane on gel) per column.
Elute DNA from each column using 10 µL nuclease free water .
Pool eluted DNA together and quantify the concentration.
Set up ligation of purified DNA to protein expression vector using the
T4 DNA LigaseNew England BiolabsCatalog #M0202
Use 1:2 insert:vector ratio to increase the chance that each insert will receive a vector.
At this stage, the ligation volume is typically 100 µL , but this can vary.
| Number of reactions | 1 | |
| 10X T4 DNA Ligase Buffer | 10 | |
| Vector (119 ng/μL) | 9 | |
| Insert (6 ng/μL) | 46 | |
| T4 DNA Ligase | 10 | |
| water | 25 |
Example ligation recipe to ligate size selected DNA into protein expression vector. All volumes in tables are in μL.
Run the ligation at Room temperature for Overnight
1h 30m
Purify the ligated DNA library.
Use a single column and elute all DNA in10 µL nuclease free water .
It is not necessary to quantify the DNA concentration.
Electroporation and library complexity quantification for ORF insertion library
Electroporation and library complexity quantification for ORF insertion library
From this point onward, it is generally acceptable to move away from expensive commercial electrocompetent cells to in-house prepared electrocompetent cells. The procedures are therefore nearly identical to the steps from go to step #10 to go to step #11 with minor changes. To make the protocol easier to follow, the steps are reproduced below with the changes incorporated.
Electroporate the ligated, ORF insertion DNA library into in-house electrocompetent cells.
Aliquot 2 * 1 mL SOC medium into microcentrifuge tubes or 30 mL universal tubes / test tubes and warm up the medium in tubes at 37 °C for at least00:30:00 .
Thaw and then mix 100 µL electrocompetent cells with the eluted DNA library in a pre-chilled microcentrifuge tube. Place the tube on ice for 00:05:00
Aliquot 55 µL DNA-competent cell mixture into a Gene Pulser/MicroPulser Electroporation Cuvettes 0.1 cm gapBio-Rad LaboratoriesCatalog #1652089
Ensure that the mixture is loaded in the gap. Place the cuvette on ice for 00:05:00
Take out the warmed up SOC medium.
Wipe down any traces of water on the cuvettes. Carry out electroporation using
Equipment
MicroPulser Electroporator
NAME
Electroporator
TYPE
Bio-Rad laboratories
BRAND
1652100
SKU
Use protocol EC1. Between each cuvette, jot down the time constants for electroporation event. The value should be between 4 to 5.9 ms. Even if the value is out-of-range, keep going.
Immediate after electroporation, add 945 µL SOC into the electroporation cuvette. Gently suspend cells by mixing up and down for 1-2 times. Transfer all suspended cells in medium into the microcentrifuge or universal tube where the medium was warmed. For samples of the same library, pool the suspended cells together.
It may be helpful to do this aseptically, but this is not strictly required. Speed is more important.
After finishing the electroporation steps for all samples, incubate the tubes at 160 rpm, 37°C, 01:00:00 for recovery.
Retrieve the recovered cells.
For each library, there should be ~ 2 mL of recovered cells. Take out 10 µL recovered cells and dilute it into 90 µL of 0.85% saline . Serially dilute it again 10-fold for 5 times. Label the tubes from A (lowest dilution) to E (highest dilution).
Spread 50 µL diluted cells onto 90 mm LB agar + KAN + TET plates labelled A to E. This is to quantify the "library complexity".
Note that the antibiotics used change here to reflect to selection of different genetic components. TET is for selecting the protein expression vector, it may change if you use a different protein expression vector. KAN is for selecting the transposon.
For the rest of the recovered cells, spread onto 2 * 145 mm LB agar + KAN + TET plates, each using ~ 1 mL of the cells. Allow the plates to dry before inverting them upside down.
Incubate all plates overnight at 37 °C
Retrieve the plates and determine library complexity.
For each library, tally the colony counts from plates A - E and create a table like below:
Expected result
| Plate | # of colonies | dilution factor | Initial number of transformants | |
| A | TMTC | 400 | - | |
| B | 108 | 4000 | 432000 | |
| C | 13 | 40000 | 520000 | |
| D | 1 | 400000 | 400000 | |
| E | 0 | 4000000 | 0 |
Example record table for calculating library complexity for the ORF insertion library.
Total number of colonies (aka library complexity) was 4.32 * 105.
Size of trimmed CDS is 767 bp.
Therefore, library fold coverage was 4.32 * 105/ ((767- 1) * 2) = 282 fold
NOTE: when calculating library fold coverage for the ORF insertion library, the formula becomes:
coverage = number of colonies / ((size of transposition window) * 2)
the size of transposition = size of the trimmed CDS -1
If the library fold coverage is > 100. Proceed. Otherwise, discard the library.
The rest of the steps including cell scrapping and midiprep are the same.
Substitution of transposon with split intein
Substitution of transposon with split intein
4h
4h
Take DNA from midipreped ORF insertion library, mix with the substitution insert in a 1:5 for library : insertion ratio and add to Golden Gate Assembly reaction.
The substitution insert is provided in excess to increase the chance that all transposon within the library is replaced.
Set up 6 * 20 µL of Golden Gate Assembly reaction in total.
Enzymes used areBbsI-HF - 1,500 unitsNew England BiolabsCatalog #R3539L , SapI - 1,250 unitsNew England BiolabsCatalog #R0569L , andT4 DNA LigaseNew England BiolabsCatalog #M0202
Note: the maximum amount of ORF insertion library DNA per 20 μL of Golden Gate reaction mix is 60 ng.
| Number of reactions | 1 | 6 | |
| 10X T4 Ligase Buffer | 2 | 12 | |
| BbsI-HF | 1 | 6 | |
| SapI | 1 | 6 | |
| T4 DNA Ligase | 1 | 6 | |
| ORF insertion library (452 ng/μL) | 0.133 | 0.798 | |
| substitution insert (42 ng/μL) | 2.6 | 15.6 | |
| water | 12.267 | 73.602 |
Example recipe for a Golden Gate substitution reaction. All volumes in tables are in μL.
Aliquot the master mix into 2 * 60 µL of reactions .
Place them on thermocyclers and run the following program:
| Step | Temperature (°C) | Time | ||
| 1 | Digestion | 37 | 3 mins | |
| 2 | Ligation | 16 | 4 mins | |
| Repeat steps 1 - 2 for 25 cycles | ||||
| 3 | Final Digestion | 37 | 30 mins | |
| 4 | Heat Inactivation | 65 | 20 mins | |
| 5 | Storage | 16 | infinite |
Thermocycler program for Golden Gate substitution reaction.
This protocol typically takes 04:00:00 .
4h
Pool all the Golden Gate Assembly reaction mixtures together and purify the final library.
Use a single column and elute all DNA in10 µL nuclease free water .
It is not necessary to quantify the DNA concentration.
Electroporation and library complexity quantification for the final library
Electroporation and library complexity quantification for the final library
Repeat go to step #21 to go to step #22.5 .
Note: if the target protein needs a reporter plasmid to manifest its function, then it is necessary to make bespoke electrocompetent cells that carry the reporter plasmid.
After the recovery step, there should be ~ 2 mL of recovered cells per library. Take 1 mL recovered cells and save as a glycerol stock.
Continue with go to step #22.6 and go to step #23 to estimate library complexity for the final library.
Screening for AND / NAND gate behavior
Screening for AND / NAND gate behavior
There is no fixed way to screen the final library. There could be different ways in which steps of enrichment or selection are conducted. Yet, in any case, the objective is to first isolate single colonies from the library that clearly show restoration of function when both the N- and C-lobes are expressed. The following steps provide a general description of what happens next.
Once single colonies that show restoration of function are obtained, they are picked into 96-well plates with medium supplemented with appropriate antibiotics. Typically, > 500 colonies are picked.
Grow the plates at 1000 rpm, 37°C, 16:00:00 (overnight) to give a saturated culture.
Dilute 2 µL overnight culture into 96-well plates with 198 µL medium supplemented with appropriate antibiotics and with 4 different combinations of inducers.
Assuming the inducer L-( )-ArabinoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #A3256 drives expression of the N-lobe and the inducer 24-DiacetylphloroglucinolCambridge BioscienceCatalog #16345 (DAPG) drives expression of the C-lobe, the 4 combinations would be
- water + DMF (solvent for DAPG)
- arabinose + DMF
- water + DAPG
- arabinose + DAPG
Grow inoculated plates at 1000 rpm, 37°C . The end time of this screening assay depends on the experiment context.
Assay the plates and identify clones that restore the original protein function when both inducers are present.
Cherry-pick and consolidate the individual wells into a new 96-well plate. Usually 93 - 195 colonies are good enough for subsequent steps.
Using a robotic liquid handler like OT-2 is strongly recommended, otherwise you risk transferring the wrong wells because this task is prone to mental exhaustion and hence mistakes.
Equipment
OT-2
NAME
Liquid handler
TYPE
Opentrons
BRAND
OT-2
SKU
Grow the consolidated plate(s) at 1000 rpm, 37°C, 16:00:00 . This plate is now the stock. Duplicate this plate and make a 96-well -80 °C glycerol stock plate by adding 100 µL bacterial culture to 50 µL 50% glycerol . Freeze immediately in -80 °C freezer
Repeat go to step #32 to go to step #33 on the isolated clones for a rigorous experiment.
Clones might behave different from the first time they are screened out. Keep only the clones that give consistent behavior.
Whole-cell PCR and sequencing to identify split sites
Whole-cell PCR and sequencing to identify split sites
Once the clones are confirmed to show consistent AND or NAND behavior, proceed to whole-cell PCR.
You only needs to amplify and sequence the region where the intein-extein CDS region join together. Both the N- or C- junction could be sequenced. In most cases the analysis is easier if the inteinC-exteinC junction is sequenced. This option is assumed for the steps below.
Design and order primers
The forward primer should anneal ~ 50 - 100 bp before the 3' end of the inteinC CDS and the reverse primer should anneal ~ 50 - 100 bp after the 3' end of the permitted transposition window.
While designing primers, estimate the maximum size possible for the PCR product.
Carry out whole-cell PCR using Taq DNA Polymerase with ThermoPol® BufferNew England BiolabsCatalog #M0267L
This is equivalent to colony PCR just that bacterial cells were obtained from liquid cultures instead of solid medium.
Assuming you have 93 candidate clones to sequence, prepare 2 tubes of the master mixes below, each in a 1.5 mL microcentrifuge tube:
| Number of reactions | 1 | 52 | |
| 10X ThermoPol Buffer | 2.5 | 130 | |
| 1mM dNTP | 5 | 260 | |
| FW primer | 0.5 | 26 | |
| RV primer | 0.5 | 26 | |
| Template bacteria | 0.5 | - | |
| Taq Pol | 0.125 | 6.5 | |
| water | 15.875 | 825.5 |
Example recipe for whole-cell PCR. All volumes in tables are in μL.
Aliquot 24 µL PCR master mix into each well of a 96-well PCR plate, then add 1 µL bacterial culture to the wells. There is no need to mix by pipetting up and down, which introduces bubbles. Seal the plate with anAluminium SealStarLabCatalog #E2796-9792 .
Run the following protocol
| Step | Temperature (°C) | Time | ||
| 1 | Initial Denaturation | 95 | 2 mins | |
| 2 | Denaturation | 95 | 15 secs | |
| 3 | Annealing | 51 | 15 secs | |
| 4 | Extension | 68 | 1 mins | |
| Repeat steps 2 - 4 for 35 cycles | ||||
| 5 | Final Extension | 68 | 5 mins | |
| 6 | Storage | 12 | infinite |
Example whole-cell PCR thermocycling program. Assumes 1 kb of maximum PCR product size and that Taq's extension speed is 1 kb /min
Resolve 2.5 µL PCR producto on agarose gel to affirm PCR is successful.
Purify the PCR product.
Elute DNA from each sample in20 µL nuclease free water .
Dilute the eluted DNA to appropriate concentrations (10 ng/μL for Source BioSciences) and send to sequencing.
Note: this step can be done manually but it is best to be facilitated using a robotic liquid handler.
Equipment
OT-2
NAME
Liquid handler
TYPE
Opentrons
BRAND
OT-2
SKU
Customize the Python scripts from our Github repository and analyze the sequencing data to identify split sites for each candidate clones. Merge split site information and protein activity data to produce an intein-bisection map.
Expected result
The intein-bisection map of β-lactamase.
Left panel, optical densities of the controls that provide the references as to how cells with or without intact β-lactamase (BLA) would grow when challenged by ampicillin. Right panel, bisection map of β-lactamase split by the split gp41-1 intein. Each vertical group of spots represents an identified split site on the x axis, aligned to the β-lactamase secondary structure (from PDB: 1ZG4) below. A total of 55 candidate strains showing AND gate behavior were characterized and sequenced to generate this map. y locations and error bars are mean and std of optical densities from independent experiments performed on three different days. Vertical dashed lines bound the permitted transposition window.