iGEM 2018 — Team UC_San_Diego

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:UC San Diego/Notebook

[![](https://static.igem.org/mediawiki/2018/2/2a/T--UC_San_Diego--nav.png)](https://2018.igem.org/Team%3AUC_San_Diego)

People

[Our Team](https://2018.igem.org/Team%3AUC_San_Diego/Team)

[Collaborations](https://2018.igem.org/Team%3AUC_San_Diego/Collaborations)

[Attributions and Sponsors](https://2018.igem.org/Team%3AUC_San_Diego/Attributions)

Application

[Product Design](https://2018.igem.org/Team%3AUC_San_Diego/Design)

[Entrepreneurship](https://2018.igem.org/Team%3AUC_San_Diego/Entrepreneurship)

[Database](https://2018.igem.org/Team%3AUC_San_Diego/Database)

Human Practices

[Human Practices Silver](https://2018.igem.org/Team%3AUC_San_Diego/Human_Practices_Silver)

[Integrated Human Practices](https://2018.igem.org/Team%3AUC_San_Diego/Human_Practices)

[Education and Public Engagement](https://2018.igem.org/Team%3AUC_San_Diego/Public_Engagement)

[Modeling](https://2018.igem.org/Team%3AUC_San_Diego/Model)

Our Project

[Our Design Philosophy](https://2018.igem.org/Team%3AUC_San_Diego/Design_Philosophy)

[Description](https://2018.igem.org/Team%3AUC_San_Diego/Description)

[Design](https://2018.igem.org/Team%3AUC_San_Diego/Applied_Design)

[Hepatocellular Carcinoma](https://2018.igem.org/Team%3AUC_San_Diego/HC)

[Protocols and Notebook](https://2018.igem.org/Team%3AUC_San_Diego/Notebook)

[Parts and Results](https://2018.igem.org/Team%3AUC_San_Diego/Results)

[Medal Criteria](https://2018.igem.org/Team%3AUC_San_Diego/Medal)

[InterLab](https://2018.igem.org/Team%3AUC_San_Diego/InterLab)

[Safety](https://2018.igem.org/Team%3AUC_San_Diego/Safety)

## Protocols

![protocol](https://static.igem.org/mediawiki/2018/b/b8/T--UC_San_Diego--protocol.png)

Gene Fragment PCR

#### Reaction mixture: (20uL)

|  |  |
| --- | --- |
| Master Mix (2X) | 10ul |
| 10uM Primer F | 1ul |
| 10uM Primer R | 1ul |
| Water | 7ul |
| Template fragment | 1ul (~10ng) |

#### PCR cycle:

|  |  |  |
| --- | --- | --- |
|  | Temp | Time |
| Initiation | 98 | 30sec |
| Cycles 30X | 98 50 (or optimal annealing temperature  72 | 5sec 30sec  20sec |
| Final | 72 | 2min |
| Hold | 4 | forever |

Gel Extraction

1. Run gels to visualize PCR products
   1. Make 2% agarose gel with EB
   2. Load all (20ul) of PCR product mixed with appropriate amount of loading buffer
   3. Remember to use big combs, skip a lane between different samples if possible when load the gel: it will make gel-cutting easier
2. Extract DNA from gels using Qiagen Gel Extraction kit
3. Determine DNA concentration using nanodrop.

Gibson Assembly

Gene fragment pool preparation (5ul total)

### Gibson1 (hMBD2):

|  |  |
| --- | --- |
| Egfp-eh | .06 uL |
| Hmbd2 | 0.5 uL |
| pET28a | 3uL |
| water | 0.5 uL |
| Master mix 2X | 5ul |

### Gibson2 (mMBD1):

|  |  |
| --- | --- |
| Egfp-em | .5ul |
| Mmbd1 | 0.5ul |
| pET28a | 3ul |
| water | 0ul |
| Master mix 2X | 5ul |

Mixed and incubate at 50 with shaking for 1hr

Cell Transformation

Cell Transformation of Gibson Assembly Samples
Positive control of Gibson Assembly - Ampicillin Resistance
Competent cell: XL10 (50uL each)

Transferred 1uL Gibson Assembly sample into competent cell, stored on ice for 40 min* Heat shock: 42 degree for 45 sec, put back onto ice
* 450uL SOC medium into each
* 37 degree incubation with shaking for ~1h
* Spread on pre-made Amp or Kana (50ug/ml) plate, overnight (8:00PM to Next day 11:00AM, 15hr)

Vector pACYC184* 37 shaker overnight

Mini Prep (Qiaprep)

1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
2. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
5. Apply 800 µl of the supernatant from step 4 to the QIAprep 2.0 spin column by pipetting.
6. Centrifuge for 30–60 s. Discard the flow-through.
7. Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.
8. Wash QIAprep 2.0 spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
9. Discard the flow-through, and centrifuge at full speed for an additional 1 min to remove residual wash buffer.
10. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.

Protein Culture

#### Starter culture:

* Add 6mL autoclaved LB into a 50mL Falcon tube, with 6uL 50mg/mL Kanamycin stock
* Pick a colony from the plate using a sterile pipet tip and put into the tube
* Incubated with shaking at 37 degree, stopped when you see the LB became cloudy (usually around 3 hours)

#### Large culture:

* Pre-heated the 2L flasks with LB at 37 degree, add 750uL 50mg/mL Kanamycin stock into each flask
* After the flasks warmed up, add 1.5mL starter culture into each flask
* Monitor the OD600 1h, 2h and more frequent when it goes above 0.2 (usually 2h30min-4h)
* Cool the flasks on ice when OD600 is approaching 0.8
* Take a gel sample with 100uL culture, centrifuged at full speed for 10min at 4 degree, discard the supernatant and resuspend the pellet in 6uL 300mM NaCl + 20mM Tris (pH = 8), add 6uL SDS-PAGE gel loading buffer
* Add IPTG into each flask until 0.5mM and change the incubator to room temperature
* Wait until the incubator has cooled, put the flasks in

E.coli harvest:* A gel sample after induction: take 100uL culture, centrifuged at full speed for 5min, discard the supernatant, resuspend the pellet in 9uL 300mM NaCl + 20mM Tris (pH = 8), +3uL GLB
* Clean two 250mL centrifuge bottles
* Centrifuge the culture in centrifuge bottles at 8000rpm for 10min at 4 degree each time, discard the supernatant each time

E.coli cell resuspension and lysis* Resuspend the pellet in ~25mL 300mM NaCl + 20mM Tris (pH = 8), remember to thoroughly pipet up and down or vortex to resuspend the pellet into a homogenous viscous solution, transfer the final solution into a 50mL Falcon tube
* Add protease inhibitor and lysozyme to the resuspension, mix thoroughly by inverting the tube several times
* Take enough liquid nitrogen to soak the tube in for ~15min

DNAse* Thaw the lysate in room temperature water bath
* Add DNAse with 75uL 1M MgCl2, 15uL 1M CaCl2, mix thoroughly by inverting the tube several times (should see lysate cluster together)
* Put the lysate in room temperature water bath for ~1h, until less viscous (1000uL pipet can easily pipet up and down)
* Take a gel sample of the lysate: 9uL lysate + 3uL GLB
* Centrifuge the lysate at 10000rpm for 1h at 4 degree, separate the supernatant immediately after centrifuge
* Take a gel sample from the supernatant: 9uL supernatant + 3uL GLB
* Resuspend the pellet in ~5mL resuspension buffer, take a gel sample from the resuspension: 9uL resuspension + 3uL GLB

Starter Culture of mMBD-eGFP, hMBD-eGFP* 6mL LB broth + 6uL 50mg/mL Kanamycin + colonies of mMBD-eGFP/hMBD-eGFP
* Incubated at 37 degree with shaking
* mMBD-eGFP starter culture completed in 3h: cloudy can barely see the toothpick
* hMBD-eGFP starter culture remained clear: probably some problems with the plate --> redo BL21 transformation

Large Culture of mMBD-eGFP (2\*750mL)* Culture + Kanamycin to final conc. Of 50ug/mL, preheated at 37 degree
* 1.5mL (1:500 dilution) starter culture added to each 750mL
* Stop incubation after ~3h30min when OD600 = 0.74, cool on ice
* A gel sample taken: 100uL medium centrifuged, pellet resuspended in 6uL resuspension buffer (300mM NaCl + 20mM Tris (8)), +6uL GLB
* 89mg IPTG (final conc. 0.5mM) added to each 750mL
* Incubated at 18 degree with shaking (8:15pm)
* Induction kept for 16h

Cell transformation of hMBD-eGFP (miniprep1)* An antibiotic free plate spread with 6uL 50mg/mL Kana diluted in 100uL solution, preheated at 37 degree
* 1uL miniprep + 50uL BL21, stored on ice for 40min
* Heat shock in 42 degree water bath for 45 sec, put back onto ice
* Add 300uL SOC medium, incubated in 37 degree heatblock with shaking for 1h, put back onto ice
* 100uL culture spread onto the Kana plate (7:45pm)
* Results: incubated for ~16h, some colonies (10-20)

Cell harvest & lysis of mMBD-eGFP* medium centrifuged in 2 250mL centrifuge bottle, each time 8000rpm, 10min, 4 degree
* pellet resuspended in 25mL resuspension buffer (300mM NaCl + 20mM Tris (8))
* ~10mg lysozyme added
* froze in liquid nitrogen

DNAse:* haw the cell lysate in water bath
* 25uL DNAse, 75uL 1M MgCl2, 15uL 1M CaCl2 added to cell lysate
* kept in water bath for 1h, less viscous
* a gel sample taken from the lysate: 6uL + 6uL GLB
* centrifuged in 2 25mL centrifuge bottle, 14500rpm, 1h, 4 degree
* a gel sample taken from the supernatant: 6uL + 6uL GLB

Ni column: new Ni column (3mL slurry)* column washed with water 3 times, resuspension buffer (300mM NaCl + 20mM Tris (8)) 3 times
* DNAse supernatant loaded, a gel sample taken from the flow through (6uL + 6uL GLB)
* column washed with resuspension buffer (300mM NaCl + 20mM Tris (8)) 3 times, a gel sample taken from the wash (6uL + 6uL GLB)
* elution 1: 6mL 25mM imidazole + 100mM NaCl + 20mM Tris (pH = 8)
* elution 2: 6mL 100mM imidazole + 100mM NaCl + 20mM Tris (pH = 8)
* elution 3: 6mL 250mM imidazole + 100mM NaCl + 20mM Tris (pH = 8)
* a gel sample taken from each of the elution (6uL + 6uL GLB)

DNA Hybridization

* Get 1ml 3X SSC from 10X SSC (300ul + 700ul water)
* Added 2ul for each complementary ssDNA and 46ul 3XSSC to 50ul

|  |  |  |
| --- | --- | --- |
| # | Forward | Reverse |
| 1 | 0\_0 | 0\_0 |
| 2 | 0\_0 | M\_0 |
| 3 | M\_0 | M\_0 |
| 4 | 0\_M | M\_0 |

* Hybridization setup: 98C for 5min, Ramp -1C/min for 70min, 4C Hold
* After reaction, each tube should have 50ul \* 4uM dsDNA = 0.2nmol DNA

DNA-protein reaction* 50ul reaction 9ul dsDNA (0.036nmol) + (+0.5nmol) 4ul mMBD-eGFP + 37ul binding buffer
* Pre-incubate mMBD-eGFP + binding buffer for 10min at RT
* Incubate for 30min at RT
* Binding samples: 10ul mixture with 2ul loading dye (loaded 10ul)

DNA only samples* Dilute 9ul dsDNA in 41ulwater (labeled "1:5"), take 10ul mixed with 2ul loading dye (loaded 10ul)

Gel: 2% agarose in 1X TAE

EMSA:

*Polyacrylamide Gel: (6%)*

3ml 30% PA (29:1) Bio-Rad
3.75mL 2X TBE
8.25mL DD water
150ul 10%APS
15ul TEMED* Wait 30min for gel to polymerize
* Pre-run with 0.5X TBE at 4 degree
* Gel 1 (wells not perfect): ladder, 2:1, 1:1, dsDNA, ssDNA (12uL each)
* Gel 2: ladder, 3:1, 5:1, dsDNA (12uL each)
* Run at 100V at 4 degree for ~1h
* Stained in 50mL 0.5x TBE + 5uL SybrSafe for 30min

*Protein-DNA interaction: (50uL each)*

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | 1:1 | 2:1 | 3:1 | 5:1 |
| mMBD-eGFP | 4 | 8 | 12 | 20 |
| Binding buffer (5x) | 10 | 10 | 10 | 10 |
| H2O | 27 | 23 | 19 | 11 |
| dsDNA | 9 | 9 | 9 | 9 |

5x binding buffer:
50mM HEPES (7.9)
15mM MgCl2
50% glycerol
5mM DTT
500mM KCl

5x gel loading buffer (no SDS):
50mM EDTA
16.5mM Tris-HCl (8)
Bromophenol blue

* Pre-incubate protein + binding buffer + H2O at RT for 10min
* + dsDNA, incubate at RT for 30min
* +12.5uL 5x gel loading buffer (no SDS)

Graphene Oxide

GO assay target 5nM, 50ul reactions

All in NEB buffer 2* Quenching assay
* GO Concentration: 0.02mg/mL
* DNA probe concentration: 50nM DNA-FAM
* Incubation time: 10min

Buffer (ph 7.9): NEB 2 (1X)* 50mM NaCl
* 10mM Tris-HCL (8.0)
* 10mM MgCl2
* 1mM DTT

Buffer (pH 7):* NEB 1 (should be available with Exo III)
* 10mM Bis-Tris-Propane-HCl
* 10mM MgCl2
* 1mM DTT

Prep:* Dilute GO in NEB 2 buffer (or 1, need comparison).
* Dilute DNA probe stock in 10mM phosphate buffer pH 7.4

Quenching (bold indicate main optimizable parameters)1. In black 96 well, add **50nM** (final conc) DNA-FAM in at least three replicates (3+ negative control)
2. To experimental groups. Add GO solutions with a final conc of **0.02mg/ml** and extra water to a total of 50ul, tap to mix (or carefully pipette up and down), incubate for **10+ min**
3. At the same time add RNA-grade water to 50ul for all negative controls
4. Read fluorescence with a excitation at 480nm, emission from 505 to 600nm

Recovery1. Keep 1+ negative control, perform the 4 steps in Quenching assay
2. Prepare serial target probe dilutions (final concentration: 0pM, 5pM, 10pM, 100pM, 500pm, 5nM, 50nM) If successful move forward to lower conc
3. Add serial probe dilutions in phosphate buffer (or NEB 2?), mix, incubate for ? mins
4. (need to confirm) if GO could be precipitate by centrifugation, centrifuge down, draw out supernatant and load to separate wells, resuspend with buffer (?), do it again

1. In a 96 clear well plate add GO solution in three replicates
2. Quenching assay: in
3. add probe solution to wells, tap to mix, incubate for 10+ min

*Protein-DNA interaction: (50uL each)*

|  |  |  |
| --- | --- | --- |
|  | 7:1 | 5:1 |
| mMBD-eGFP | 30 | 20 |
| Binding buffer (5x) | 10 | 10 |
| H2O | 1 | 11 |
| dsDNA | 9 | 9 |

Quenching Assay

#### DNA probe

Required buffer: (self making or borrowing the stocks) pH is crucial* 10mL 10mM sodium phosphate pH 7.4 from 1M sodium phosphate
* NEB II buffer (ask whether they have it, if not prepare a 5X stock)
50mM NaCl
10mM Tris-HCL (usually they have pH 8.0)
10mM MgCl2
1mM DTT
pH 7.9

Required equipment:* 96 well fluorescent plate, borrowed from Zhang's lab or the lab with Tecan, black/non black both are fine (transparent will be better if you can get one)
* Tecan Reader

Procedures:1. Get access to fluorescent labeled DNA order info from IDT: order number 15387329 (should have 3, they are in one of the envelopes on the desk)
2. Follow instruction to dilute each DNA sample to 100uM with 10mM phosphate pH 7.4,
3. Prepare 200ul 10pM, 1nM, 100nM for each fluorescent DNA with NEB II buffer. Take a bit 100uM and make serial dilutions with dilution factors not bigger than 10 (10uM, 1uM, 100nM…)
4. Take 10ul 2mg/ml GO dispersion (in the right top drawer of our cleanest bench) and dilute to 0.02mg/ml in NEB II buffer by pipette mixing, invert tube couple times before use
5. In 96 well plate, add 25ul fluorescent DNA dilutions in four replicates (see sample plate map). 0nM DNA = neat NEB II buffer. 250pM, 5nM follow same setup as 10pM (with+without GO)
6. In "-with GO" wells, add 25ul 0.02mg/ml GO solution; In "-without GO" wells, add 25ul NEB II buffer
7. Shake at Tecan reader and record fluorescence at 5min, 10min, 15min with excitation at 480nm, emission from 520nm

Slot Blot

Input: 100ng, about 6 pmol
2ul each ssDNA, 46 3X SSC (4uM)
load 2ul each slot (8pmol)

Procedures:
Each membrane: 12 slots, (2cm \*2cm)
Condition: (Crosslink/not)\* 0.05X, 0.005
X (4 total)

Soak blot paper with 6X SSC for 10min
Wash each slot with 0.4mL TE, wait for dry
Pipette 2ul to each slot, wait for dry
Wash each slot with 0.4ml 2X SSC, wait for dry

Crosslink or not
Soak membrane in PBS-T for 10min
Incubate in blocking buffer for 50min (2.5g milk in 50mL PBS-T
Dilute mMBD-eGFP in PBS-T (10mL for each), incubate overnight at 4

Wash membrane with PBS-T twice
Incubate with AntiHis-HRP

## Notebook

We have also documented our overall lab activities through several notebooks.


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:UC San Diego/Experiments

[![](https://static.igem.org/mediawiki/2018/2/2a/T--UC_San_Diego--nav.png)](https://2018.igem.org/Team%3AUC_San_Diego)

People

[Our Team](https://2018.igem.org/Team%3AUC_San_Diego/Team)

[Collaborations](https://2018.igem.org/Team%3AUC_San_Diego/Collaborations)

[Attributions and Sponsors](https://2018.igem.org/Team%3AUC_San_Diego/Attributions)

Application

[Product Design](https://2018.igem.org/Team%3AUC_San_Diego/Design)

[Entrepreneurship](https://2018.igem.org/Team%3AUC_San_Diego/Entrepreneurship)

[Database](https://2018.igem.org/Team%3AUC_San_Diego/Database)

Human Practices

[Human Practices Silver](https://2018.igem.org/Team%3AUC_San_Diego/Human_Practices_Silver)

[Integrated Human Practices](https://2018.igem.org/Team%3AUC_San_Diego/Human_Practices)

[Education and Public Engagement](https://2018.igem.org/Team%3AUC_San_Diego/Public_Engagement)

[Modeling](https://2018.igem.org/Team%3AUC_San_Diego/Model)

Our Project

[Our Design Philosophy](https://2018.igem.org/Team%3AUC_San_Diego/Design_Philosophy)

[Description](https://2018.igem.org/Team%3AUC_San_Diego/Description)

[Design](https://2018.igem.org/Team%3AUC_San_Diego/Applied_Design)

[Hepatocellular Carcinoma](https://2018.igem.org/Team%3AUC_San_Diego/HC)

[Protocols and Notebook](https://2018.igem.org/Team%3AUC_San_Diego/Notebook)

[Parts and Results](https://2018.igem.org/Team%3AUC_San_Diego/Results)

[Medal Criteria](https://2018.igem.org/Team%3AUC_San_Diego/Medal)

[InterLab](https://2018.igem.org/Team%3AUC_San_Diego/InterLab)

[Safety](https://2018.igem.org/Team%3AUC_San_Diego/Safety)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:UC San Diego/Results

[![](https://static.igem.org/mediawiki/2018/2/2a/T--UC_San_Diego--nav.png)](https://2018.igem.org/Team%3AUC_San_Diego)

People

[Our Team](https://2018.igem.org/Team%3AUC_San_Diego/Team)

[Collaborations](https://2018.igem.org/Team%3AUC_San_Diego/Collaborations)

[Attributions and Sponsors](https://2018.igem.org/Team%3AUC_San_Diego/Attributions)

Application

[Product Design](https://2018.igem.org/Team%3AUC_San_Diego/Design)

[Entrepreneurship](https://2018.igem.org/Team%3AUC_San_Diego/Entrepreneurship)

[Database](https://2018.igem.org/Team%3AUC_San_Diego/Database)

Human Practices

[Human Practices Silver](https://2018.igem.org/Team%3AUC_San_Diego/Human_Practices_Silver)

[Integrated Human Practices](https://2018.igem.org/Team%3AUC_San_Diego/Human_Practices)

[Education and Public Engagement](https://2018.igem.org/Team%3AUC_San_Diego/Public_Engagement)

[Modeling](https://2018.igem.org/Team%3AUC_San_Diego/Model)

Our Project

[Our Design Philosophy](https://2018.igem.org/Team%3AUC_San_Diego/Design_Philosophy)

[Description](https://2018.igem.org/Team%3AUC_San_Diego/Description)

[Design](https://2018.igem.org/Team%3AUC_San_Diego/Applied_Design)

[Hepatocellular Carcinoma](https://2018.igem.org/Team%3AUC_San_Diego/HC)

[Protocols and Notebook](https://2018.igem.org/Team%3AUC_San_Diego/Notebook)

[Parts and Results](https://2018.igem.org/Team%3AUC_San_Diego/Results)

[Medal Criteria](https://2018.igem.org/Team%3AUC_San_Diego/Medal)

[InterLab](https://2018.igem.org/Team%3AUC_San_Diego/InterLab)

[Safety](https://2018.igem.org/Team%3AUC_San_Diego/Safety)

## Results

## Plasmid construction

Two basic constructs and two improved constructs were assembled in this project. Each basic construct contains a single MBD binding domain coupled with eGFP and avi-tag for signal detection. Each improved construct contains a human MBD domain and a mouse MBD domain, aiming to improve DNA affinity while maintaining good expression at the same time. More information about the constructs could be referred to the design page.

A. Basic constructs: mMBD-eGFP and hMBD-eGFP
Pre-digested pET28A vector (NcoI and XhoI) was obtained from Bottini’s lab in UCSD. The vector was analyzed with agarose gel (Figure 1) with 2-log ladder (same below unless specified). The digested vector was then extracted with Qiagen Gel Extraction Kit and eluted. All DNA concentrations were quantified by Nanodrop 2000. Concentrations of the vector were summarized in Table 1. To increase the concentration of pET28A, SpeedVac was applied to the sample to reach a final concentration of 15ng/ul.

Gblocks containing mouse MBD (mMBD), human MBD (hMBD), and enhanced green fluorescent protein (eGFP) sequences with Avi-tag and thrombin site (the whole gblock is called “eGFP” for simplicity) were purchased separately. Expected sizes for fragments were indicated in Table 2. PCR reactions were carried out with gblocks so that: 5’end of mMBD and hMBD have 20nt overlap with the linearized vector and 3’end have 20nt overlap with eGFP; 3’end of eGFP has 20nt overlap with the linearized vector. (all primer sequences: supplementary table). PCR overlap products were subsequently analyzed with agarose gel and extracted (Table 3, Figure2). A PCR extension was then carried out to combine mMBD, hMBD with eGFP, respectively. (Figure 3)

Gibson assembly was subsequently carried out with linearized pET28A and hMBD-eGFP; Restriction cloning was carried out with linearized pET28A and mMBD-eGFP using NcoI and XhoI. Clones were sub-cultured overnight and miniprepped with Qiagen kit. Plasmids were sequenced to confirm correct assembly (supplementary figure).

![](https://static.igem.org/mediawiki/2018/c/cf/T--UC_San_Diego--fig1.png)
![](https://static.igem.org/mediawiki/2018/3/38/T--UC_San_Diego--fig2.png)

##### Figure 1. gel image of pre-digested pET28A.

|  |  |  |
| --- | --- | --- |
| Vector | pET28 (S1) | pET28 (S2) |
| Expected size | About 5.2kb | |
| Concentration (ng/ul) | 2.2 | 2.0 |

##### Table 1. Concentrations of vector samples extracted from agarose gel.

![](https://static.igem.org/mediawiki/2018/2/23/T--UC_San_Diego--fig3.png)

##### Figure 2. Gel image of overlap PCR products. Sizes of gblocks are: mMBD, 216bp; hMBD, 228bp; eGFP, 792bp.

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| construct | mMBD | hMBD | eGFP | mMBD-eGFP | hMBD-eGFP |
| Size (bp) | 216 | 228 | 792 | About 1100 | About 1100 |

##### Table 2. summary of sizes of gblocks.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| PCR extension substrate | hMBD-eGFP overlap | mMBD-eGFP overlap | eGFP-MBD overlap | eGFP-mMBD overlap |
| Concentration (ng/ul) | 21.7 | 24.4 | 17.1 | 19.8 |
| A260/A280 | 1.79 | 1.9 | 1.84 | 1.82 |

##### Table 3. summary of extracted overlap PCR products.

![](https://static.igem.org/mediawiki/2018/c/c8/T--UC_San_Diego--fig4.png)

##### Figure 3. gel image of PCR extension product. Two replicates were combined into one extraction process and the concentrations of extracted product are: hMBD-eGFP, 10ng/ul; mMBD-eGFP, 33.7ng/ul.

### A. Improved construct: hm-avi and hm-HRP

gblock fragments of hm-avi and hm-HRP (see design page for construct details) were purchased from IDT DNA. Primers were designed to amplify the constructs so that they contain NcoI and XhoI overhang. Amplified constructs and pET28A vectors were subsequently digested (Figure 4), ligated, and cloned. Sequencing results were provided in the supplementary section.

![](https://static.igem.org/mediawiki/2018/e/e1/T--UC_San_Diego--fig5.png)

##### Figure 4. gel image of digested pET28A and hm-HRP.

### Recombinant protein expression and quantification

All constructs were induced with IPTG in E.coli BL-21 and initially purified with Ni-NTA column. Variants except mMBD-eGFP required further purification for higher purity. In this project, hm-avi and hm-HRP proteins were purified by SEC column. The purified proteins were then quantified by one of the two methods: two basic constructs were quantified by OD 280 via Nanodrop 2000; two improved constructs were quantified by Fast Protein Liquid chromatography (FPLC).

The constructed plasmids with their sequences confirmed was transformed into E.coli strain BL-21 (DE3) for protein expression. A starter culture containing 6mL autoclaved LB broth and 50ug/mL Kanamycin was grown for each construct. The incubation at 37 degree generally took around 3 hours for the culture to become cloudy. Then, 1.5 mL starter culture was used to grow a 750mL large culture (1:500 dilution). The incubation at 37 degree was stopped when OD600 was around 0.8. The culture was then cooled on ice and 100uL of it was taken as the reference before IPTG induction. The protein expression was induced with 0.5mM IPTG at 18 degree for 16 hours with shaking. 100uL of culture was taken after the induction to assess the expression of the protein. The E.coli cells were harvested by centrifuging the culture at 8000rpm, 4 degree for 10min each time. Each pellet was resuspended in 12mL resuspension buffer (300mM NaCl + 20mM Tris (pH =8)). The E.coli cells were then lysed by adding ~5mg lysozyme and fast freezing in liquid nitrogen for 15min.

5uL DNAseI, 15uL 1M MgCl2 and 3uL 1M CaCl2 were added into thawed E.coli cell lysate to digest the DNA. The lysate was left in room-temperature water bath for 1 hour until it became less viscous. 5uL lysate was taken as the reference before centrifuging for separation. The soluble and insoluble fractions of the lysate was separated by centrifuging at 14500rpm, 4 degree for 1h, and 5uL supernatant was taken to assess the solubility of the protein.

Purification of the recombinant protein generally took use of the His-tag at the end. 1mL Ni-NTA agarose equilibrated with resuspension buffer (300mM NaCl + 20mM Tris (pH = 8)) was used for each construct. The supernatant after centrifugation was loaded, the column was then washed with 3 column volumes of resuspension buffer. For hMBD-eGFP and mMBD-eGFP, 5 4mL elutions containing imidazole gradient was performed (50mM, 100mM, 150mM, 200mM and
250mM). For hm-Avi and hm-HRP, 3 6mL elutions containing wider imidazole gradient was performed (10mM, 100mM, 250mM).

TGX stain-free gel was run to check the expression, solubility, and Ni column purification of each construct. Unstained protein ladder from Bio-Rad was used for all the stain-free gels. For hMBD-eGFP, the expression of the protein is not significant according to the gel (Figure 5). Neither could the corresponding band be seen in the lysate, supernatant and Ni column elutions. However, the fluorescence of eGFP was clearly observed in at least two of the Ni column elutions. Thus, the gel was transferred onto a nitrocellulose membrane and blotted with anti-His antibody labeled with HRP. The band of expected size was observed in Ni elution 2 and 3 after HRP substrate was applied. However, further purification was required to remove the impurity (Figure 6). For mMBD-eGFP, the expression of the protein is not significant according to the gel and its solubility is also hard to determine, but the band of expected size was observed in Ni elution 2 and 3 (Figure 7). From the gel, Ni elution 3 is purer, so it is used for EMSA later. To get rid of the possible effect of imidazole on a later experiment, the mMBD-eGFP Ni elution 3 was dialyzed in 3000 MWCO with 500mL 150mM NaCl + 20mM Tris (7.3) overnight. The concentration of the sample after dialysis was determined by Nanodrop to be 7.88uM. For hm-Avi, the difference between before and after induction clearly shows the expression of the protein, but it seems not all the protein in the supernatant bound to the Ni column, and Ni elution 2 contains the protein bound to the column (Figure 8). Concerned about the purity of the protein from the gel, the Ni elution 2 was concentrated using 3000 MWCO ultracentrifuge from Satorius to around 300uL and loaded onto Size Exclusion Column 650 from GE. The buffer used for the column was 150mM NaCl + 20mM Tris (pH = 7.3). 4 peaks were observed on the chromatography (Figure 9), and the fractions were loaded onto a TGX stain-free gel with the load to see their content. From the gel, fraction 6 of gel filtration is shown to contain the band of hm-Avi (Figure 10), and Western Blot with anti-His labeled with HRP confirms that (Figure 11). Th concentration of the fraction was determined directly from the chromatography to be 3.21uM, and Nanodrop gave pretty similar results. The fraction was directly used for later experiments. For hm-HRP, the expression of the protein is not significant according to the gel, and it’s also hard to determine its solubility and presence in Ni column elutions (Figure 12). But from Western Blot of anti-His labelled with HRP, it can be observed that some protein even though not a lot is in Ni elution 2 (Figure 13). Thus, the Ni elution 2 was concentrated using 5000 MWCO ultracentrifuge from Satorius to around 300uL and loaded onto size exclusion column 650. The same buffer was used. 3 peaks were observed on the chromatography (Figure 14), and the fractions were loaded onto an SDS-PAGE gel with a load to see their content. The gel was then blotted with anti-His labeled with HRP. The Blot shows that fraction 3 contains hm-HRP (Figure 15). The chromatography calculates the concentration of fraction 3 to be only 1.57uM, so it was concentrated using 10000 MWCO ultracentrifuge from Satorius to around 300uL. The concentration was once again determined by Nanodrop to be 3.29uM. The fraction after concentration was used for later experiments.

![](https://static.igem.org/mediawiki/2018/8/80/T--UC_San_Diego--fig6.png)
![](https://static.igem.org/mediawiki/2018/c/c7/T--UC_San_Diego--fig7.png)

##### Figure 5. TGX stain-free gel of hMBD-eGFP with Unstained Protein Ladder from Bio-Rad. The expected size of hMBD-eGFP is 39 kDa.

![](https://static.igem.org/mediawiki/2018/8/85/T--UC_San_Diego--fig8.png)

##### Figure 6. Anti-His labelled with HRP of hMBD-eGFP. The expected size of hMBD-eGFP is 39 kDa.

![](https://static.igem.org/mediawiki/2018/2/2c/T--UC_San_Diego--fig9.png)

##### Figure 7. TGX stain-free gel of mMBD-eGFP. The expected size of mMBD-eGFP is 39 kDa.

![](https://static.igem.org/mediawiki/2018/5/5f/T--UC_San_Diego--fig10.png)

##### Figure 8. TGX stain-free gel of hmAvi. The expected size of hmAvi is 21 kDa.

![](https://static.igem.org/mediawiki/2018/b/b9/T--UC_San_Diego--fig11.png)

##### Figure 9. Size Exclusion Chromatography of hmAvi Ni elution 2. Blue line is the absorbance at 280nm. Fraction 3, 5, 6 and 7 were later loaded onto a TGX stain-free gel with the load.

![](https://static.igem.org/mediawiki/2018/1/11/T--UC_San_Diego--fig12.png)

##### Figure 10. TGX stain-free gel of hmAvi gel filtration load and fraction 3, 5, 6, 7. The expected size of hmAvi is 21 kDa.

![](https://static.igem.org/mediawiki/2018/9/97/T--UC_San_Diego--fig13.png)

##### Figure 11. Anti-His labelled with HRP of hmAvi gel filtration load and fraction 3, 5, 6, 7. The expected size of hmAvi is 21 kDa.

![](https://static.igem.org/mediawiki/2018/d/df/T--UC_San_Diego--fig14.png)

##### Figure 12. TGX stain-free gel of hmHRP. The expected size of hmHRP is 54 kDa.

![](https://static.igem.org/mediawiki/2018/3/32/T--UC_San_Diego--fig15.png)

##### Figure 13. Anti-His labelled with HRP of hmHRP Ni elution samples. The expected size of hmHRP is 54 kDa.

![](https://static.igem.org/mediawiki/2018/c/ca/T--UC_San_Diego--fig16.png)

##### Figure 14. Size Exclusion Chromatography of hmHRP Ni elution 2. The blue line is the absorbance at 280nm. Fraction 3, 4, and 5 were later loaded onto an SDS-PAGE gel with the load.

![](https://static.igem.org/mediawiki/2018/4/47/T--UC_San_Diego--fig17.png)

##### Figure 15. Anti-His labelled with HRP of hmHRP gel filtration load and fraction 3, 4, 5. The expected size of hmHRP is 54 kDa.

### MBD variants validation

MBD constructs were characterized by electrophoretic mobility shift assay (EMSA). In this method, protein and DNA will be pre-incubated and run on a native polyacrylamide gel. If protein binds to DNA, DNA will be slowed while running; at the same time, a “retardation band” will form when the gel is visualized with DNA-specific dye. DNA not incubated with the proteins was run along the same gel as standards

Three constructs including mMBD-eGFP, hm-avi, and hm-HRP were characterized in this project. 24 bp primers containing two methylation sites in close proximity were chosen as the target to test protein-DNA binding, in order to characterize the specificity of different protein constructs. To be more clinically relevant, one of the methylation sites on the primer was cg10428836, which was shown to have significant methylation level change in hepatocellular carcinoma (HCC). The other methylation site, by contrast, has little known clinal relevance. Forward and reverse primers were pre-hybridized to dsDNA before the assay; in the figures, primers were specially labeled as like “F 0\_0”, which indicates that it was a forward primer with no methylation on both sites “0”. Another example will be “R M\_0”, which stands for the reverse primer with a site methylated near 5’ end “M” (this methylation site is cg10428836).

Pre-hybridization was performed for: no methylation (both 0\_0), hemi-methylation (0\_0 + M\_0), symmetrical methylation (M\_0, M\_0), and unsymmetrically methylation (0\_M + M\_0). A good protein construct will only bind to symmetrically methylated DNA but not hemi-methylated nor unsymmetrically methylated ones; latter will be the noises for our device.

Among the tested construct, mMBD-eGFP showed hints of protein-DNA interaction while it was not decisively conclusive for a confirmed binding (supplementary figure). hm-Avi (Figure 16) was shown to bind to not symmetrically methylated DNA. One possible reason was that the concentration of hm-Avi was too high. Lowering the protein concentration in the future could potentially increase the specificity for this protein

By contrast, Hm-HRP protein demonstrated a good specificity toward symmetrically methylated DNA (Figure 17). To confirm this result, Hm-HRP on the same gel was transferred to nitrocellulose membrane and subsequently probed with anti-His labeled HRP. The membrane was then visualized with Western Femto substrate (Figure 18) to show that Hm-HRP was present in the retardation band

In conclusion, mMBD-eGFP and hm-avi protein require further validation to confirm their binding to methylated DNA. Hm-HRP is fully validated and suitable as the methylated DNA detector for our device.

![](https://static.igem.org/mediawiki/2018/6/67/T--UC_San_Diego--fig18.png)

##### Figure 16. retardation bands were present in both negative controls and experimental condition (lane 3).

![](https://static.igem.org/mediawiki/2018/b/bd/T--UC_San_Diego--fig19.png)

##### Figure 17. retardation bands were present only symmetrically methylated DNA probes (lane 3), suggesting its specificity.

![](https://static.igem.org/mediawiki/2018/d/d4/T--UC_San_Diego--fig20.png)

##### Figure 18. Hm-HRP was transferred and visualized with anti-His antibody. Hm-HRP/DNA complex was confirmed by the presence of protein signal at lane 3.

### Characterization of Graphene oxide (GO) platform

Two steps of graphene oxide method were characterized in this project: 1. GO quenched fluorescent dye labeled DNA by hydrophobic interactions and fluorescent resonance energy transfer (FRET). 2. Fluorescence was recovered when complementary target DNA was introduced, forming duplex with fluorescent probe. Exonuclease III amplification strategy was also characterized in this section (See design pages for more details).

To provide proof-of-concept evidence of GO platform, we set up multiple small-scale reactions in 96 well plates and monitored fluorescence with Tecan Plate Reader. NEB II buffer (pH=7.9) was the solvent environment for all experiments and measurements. This was because FAM (fluorescent dye) used in the DNA probe would lose activity when pH was under 7; Exonuclease III had optimal activity at pH =7, but retained 75% activity in NEB II.

Serial dilutions of fluorescent DNA probe in NEB II (Sequences provided in supplementary) were first performed to achieve a concentration of 100nM. 0.02mg/mL GO solution was diluted from 2mg/mL stock (Sigma-Aldrich). A master mix was prepared with equal amount of DNA probe and GO solution, mixed, and subsequently transferred to 96 well plates. Fluorescent quenching was monitored and presented in Figure 19. Quenching efficiency was calculated and presented in Figure 20, which reached above 97% in 20 minutes.

In a separate trial, procedures mentioned above was repeated and the quenching was allowed to proceed for 20 minutes. 50pM DNA probe (R 0\_0, see section 3) diluted in NEB II buffer was then added, followed by addition of 20U exonuclease III. Figure 21 demonstrated that addition of both target DNA and exonuclease III recovered more fluorescence compared to negative controls.

Role of exonuclease III in signal amplification was also briefly investigated by comparing samples with and without the enzyme. In low concentration of DNA (5pM), samples without adding exonuclease failed to recover fluorescent signal (Figure 22); samples with exonuclease III exhibited a similar kinetic curve compared to Figure 21, demonstrating its importance for our device to detect low concentrations of ctDNA.

##### Graphene oxide is able to quench fluorescent DNA probe in 20min

![](https://static.igem.org/mediawiki/2018/0/05/T--UC_San_Diego--fig21.png)

##### Figure 19. n=3. 50ul 100nM fluorescent DNA probe was mixed with 50ul 0.02mg/ml GO.

![](https://static.igem.org/mediawiki/2018/7/73/T--UC_San_Diego--fig22.png)

##### Figure 20. Quenching efficiency was calculated by dividing each fluorescent signal with control fluorescence (108891). Quenching did not start at 0% probably because the kinetics were too fast to capture at the beginning.

Addition of target DNA can recover fluorescence in 60min

![](https://static.igem.org/mediawiki/2018/f/fd/T--UC_San_Diego--fig23.png)

##### Figure 21. similar as below, full kinetics were probably too fast to capture. Although background existed when only exonuclease was present, its signal was much lower than the one with target DNA

Addition of exonuclease enhance fluorescent recovery

![](https://static.igem.org/mediawiki/2018/2/29/T--UC_San_Diego--fig25.png)

### Supplementary information

A. Supplementary figures and tables mentioned in lab result pages

![](https://static.igem.org/mediawiki/2018/2/29/T--UC_San_Diego--fig25.png)

##### Figure 1. pET28A vector digested with NcoI and XhoI.

![](https://static.igem.org/mediawiki/2018/d/d3/T--UC_San_Diego--supp2.png)

##### Figure 2. Sequences of basic constructs: mMBD-eGFP and hMBD-eGFP; please refer to parts BBa\_K2881003 and BBa\_K2881004 to access sequences.

![](https://static.igem.org/mediawiki/2018/9/98/T--UC_San_Diego--supp3.png)

##### Figure 3. Sequencing results of basic constructs in pET28a: mMBD-eGFP and hMBD-eGFP

![](https://static.igem.org/mediawiki/2018/1/1e/T--UC_San_Diego--supp4.png)

##### Figure 4. sequences of improved constructs: hm-avi and hm-HRP

![](https://static.igem.org/mediawiki/2018/1/1b/T--UC_San_Diego--supp5.png)

##### Figure 5. Sequencing results of improved constructs in pET28a: hm-avi and hm-HRP

![](https://static.igem.org/mediawiki/2018/f/f1/T--UC_San_Diego--supp6.png)

##### Figure 6. EMSA result for mMBD-eGFP. Unlike hm-avi and hm-HRP, a different ladder was used with the smallest size of 100bp. Although no retardation band was observed, DNA-MBD binding was likely happen because: 1. DNA band became thinner in symmetrically methylated substrate. 2. More “streaks” were present in the same lane. A more well-crafted gel may be necessary to visualize mMBD-eGFP/DNA binding events; internal DNA control could also be used in the future to confirm that the DNA band becomes thinner.

![](https://static.igem.org/mediawiki/2018/3/3f/T--UC_San_Diego--supp7.png)

##### Table 1. sequences of primers used in this project

### BirA biotin ligase was cloned and expressed for building a versatile MBD detection platform

One important consideration we had in our BioBrick design was to make constructs as versatile as possible for different contexts. For example, detecting DNA methylation in different settings may prefer different detection methods such as colorimetric and fluorescent assays. Biotin has been applied to fulfill this need as it could be recognized by streptavidin in extremely high affinity. Many established methods have been developed to attach biotin to proteins. Here we chose to introduce avi-Tag in our construct, which could be specifically biotinylated by BirA biotin ligase; the enzymatic reaction happens in mild condition, enabling maximum preservation of MBD binding activity compared to traditional chemical treatment.

In addition to adding avi-Tag in most of our constructs, the major step we committed to building a versatile biotin detection platform for our construct is to efficiently express BirA in E.Coli BL-21. In brief, BirA gblock fragment was purchased from IDT DNA and cloned into pCDF-dueT via restriction cloning with BamH1 and SaI1. In Figure 8 we demonstrated that BirA could be expressed in high yield and purified with Ni-NTA column. Key parameters of protein expression/purification were shown in Table 2. Through this data, we aim to provide support for future IGEM projects with avi-Tag built-in constructs.

![](https://static.igem.org/mediawiki/2018/7/73/T--UC_San_Diego--supp8.png)

##### Figure 7. vector map for pCDF-Duet.

![](https://static.igem.org/mediawiki/2018/9/9d/T--UC_San_Diego--supp9.png)

##### Figure 8. gel image for BirA purification

![](https://static.igem.org/mediawiki/2018/0/09/T--UC_San_Diego--supp10.png)

##### Table 2. Key parameters for BirA expression/Nickel column purification.

## Parts

[Check out our parts here.](http://parts.igem.org/Part%3ABBa_K2881006#MBD-HRP_fusion)