iGEM 2018 — Team BFSUICC-China

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# Team:BFSUICC-China/Notebook

**BFSUICC-China**

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[Parts](https://2018.igem.org/Team%3ABFSUICC-China/Parts) [BasicParts](https://2018.igem.org/Team%3ABFSUICC-China/Basic_Part) [CompositeParts](https://2018.igem.org/Team%3ABFSUICC-China/Composite_Part)

[Human Practices](https://2018.igem.org/Team%3ABFSUICC-China/Human_Practices)

#

# Construct the circuit

#### MAKING LB AGAR PLATES

> 1.Add 250 mL of dH2O to a graduated cyclindar.
>
> 2.Weigh out,： 5.0 g tryptone；2.5 g yeast extract；5.0 g NaCl；7.5 g agar
>
> 3.Mix powder well to bring into solution
>
> 4.Add dH2O to total volume of 500 mL and transfer to 1 L flask
>
> 5.Put on stirring hot plate and heat to boil for 1 min while stirring.
>
> 6.Transfer to 1 L pyrex jar and label with autoclave tape.
>
> 7.Autoclave at liquid setting for 20 minutes in a basin making sure to loosen top
>
> 8.Let agar cool

##### Pouring the Plates

> 1.Make sure bench has been sterilize
>
> 2.Pour a thin layer (5mm) of LB Agar (~10mL) into each plate being careful to not lift the cover off excessively
>
> 3.Swirl plate in a circular motion to distribute agar on bottom completely.
>
> 4.Store plates in plastic bags in fridge with: name, date and contents (note any additive).

#### BACTERIAL TRANSFORMATION

> 1.Take competent cells out of -80°C and thaw on ice
>
> 2.Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator.
>
> 3.Resuspend DNA in selected wells in the Distribution Kit with 10µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
>
> 4.Mix 10 μl of DNA into 50 μL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times.
>
> 5.Incubate the competent cell/DNA mixture on ice for 30 mins.
>
> 6.Heat shock each transformation tube by placing the bottom 2/3 of the tube into a 42°C water bath for 60 secs.
>
> 7.Put the tubes back on ice for 2 min.
>
> 8.Add 450 μl LB (without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min.
>
> 9.Plate some or all of the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic.
>
> 10.Incubate plates at 37°C overnight.

#### INCUBATE BACTERIAL CULTURE OVERNIGHT

> 1.Prepare liquid LB.
>
> For example, to make 400 mL of LB, weigh out the following into a 500 mL glass bottle: 4 g NaCl 4 g Tryptone 2 g Yeast Extract ddH2O to 400 mL
>
> 2.When ready to grow your culture, add liquid LB to a tube or flask and add the appropriate antibiotic to the correct concentration (see table below).
>
> 3.Using a sterile pipette tip or toothpick, select a single colony from your LB agar plate.
>
> 4.Drop the tip or toothpick into the liquid LB + antibiotic and swirl.
>
> 5.Loosely cover the culture with sterile aluminum foil.
>
> 6.Incubate bacterial culture at 37°C for 12h in a shaking incubator.
>
> 7.After incubation, check for growth, which is characterized by a cloudy haze in the media.

![](https://static.igem.org/mediawiki/2018/1/16/T--BFSUICC-China--Antibiotic.png)

#### EXTRACT PLASMID DNA

> 1.Pellet 4 ml bacterial overnight culture by centrifugation at 13000 rpm for 3 min at room temperature.
>
> 2.Resuspend pelleted bacterial cells in 250μl Buffer P1 and transfer to a microcentrifuge tube.
>
> 3.Add 250μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear.
>
> 4.Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 6 times.
>
> 5.Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
>
> 6.Apply the supernatant from step 5 to the QIAprep spin column by pipetting. Centrifuge for 30–60 s and discard the flow-through
>
> 7.Wash the QIAprep spin column by adding 0.5 ml Buffer PB.
>
> 8.Centrifuge for 60 s and discard the flow-through.
>
> 9.Wash the QIAprep spin column by adding 0.75 ml Buffer PE. Centrifuge for60 s and discard the flow-through.
>
> 10.Centrifuge for 1 min to remove residual wash buffer.
>
> 11.Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50μl water to the center of the QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

#### OPTIMIZING RESTRICTION ENDONUCLEASE REACTIONS

> PstⅠ-HF
>
> Reaction Condition CutSmart Buffer, 37℃
>
> SpeⅠ
>
> Reaction Condition CutSmart Buffer, 37℃. Heat inactivation: 80℃ 20min
>
> XbaⅠ
>
> Reaction Condition CutSmart Buffer, 37℃. Heat inactivation: 65℃ 20min
>
> EcoRⅠ
>
> Reaction Condition CutSmart Buffer, 37℃. Heat inactivation: 65℃ 20min

![](https://static.igem.org/mediawiki/2018/3/31/T--BFSUICC-China--Restriction_digestion.png)

#### AGAROSE GEL ELECTROPHORESIS

##### Pouring a Standard 1% Agarose Gel:

> 1.Measure 1 g of agarose.
>
> 2.Mix agarose powder with 100 mL 1xTAE in a microwavable flask. See TAE Recipe.
>
> 3.Microwave for 1-3 min until the agarose is completely dissolved.
>
> 4.Let agarose solution cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins.
>
> 5.Pour the agarose into a gel tray with the well comb in place.
>
> 6.Place newly poured gel at 4 °C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely solidified.

##### Loading Samples and Running an Agarose Gel:

> 1.Add loading buffer to each of your DNA samples.
>
> 2.Once solidified, place the agarose gel into the gel box (electrophoresis unit).
>
> 3.Fill gel box with 1xTAE until the gel is covered.
>
> 4.Carefully load a molecular weight ladder into the first lane of the gel.
>
> 5.Carefully load your samples into the additional wells of the gel.
>
> 6.Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage.
>
> 7.Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
>
> 8.Using any device that has UV light, visualize your DNA fragments. The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel.

![](https://static.igem.org/mediawiki/2018/e/ef/T--BFSUICC-China--_Electrophoresis_Machine.jpg)

#### EXTRACTION THE DNA FRAGMENT FROM THE AGAROSE GEL

> 1.Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
>
> 2.Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 µl).
>
> 3.Incubate at 50°C for 10 min (or until the gel slice has completely dissolved).To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.
>
> 4.After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
>
> 5.Add 1 gel volume of isopropanol to the sample and mix.
>
> 6.Place a QIAquick spin column in a provided 2 ml collection tube.
>
> 7.To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min.
>
> 8.Discard flow-through and place QIAquick column back in the same collection tube.
>
> 9.Recommended: Add 0.5ml of Buffer QG to QIAquick column and centrifuge for1min.
>
> 10.To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.
>
> 11.Discard the flow-through and centrifuge the QIAquick column for an additional 1min at 13,000 rpm.
>
> 12.Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
>
> 13.To elute DNA, add 50µl water (pH7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1min. Alternatively, for increased DNA concentration, add 30µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.
>
> 14.If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

#### T4 LIGASE

> 1.Set up the following reaction in a microcentrifuge tube on ice.
>
> 2.Gently mix the reaction by pipetting up and down and microfuge briefly.
>
> 3.Incubate at 16°C overnight or room temperature for 10 minutes.
>
> 4.Heat inactivate at 65°C for 10 minutes.
>
> 5.Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

![](https://static.igem.org/mediawiki/2018/8/81/T--BFSUICC-China--Ligase.png)

#### AGAROSE GEL ELECTROPHORESIS（checking）

> we run the electrophoresis again to check the base pair is correct or not, thus we could know our ligation is successful or not.

#

#

# Test the function of parts and circuit

###

#####

|  |
| --- |
| Plate Reading (Relative and Absolute Fluorescence)Day 1 : Pick 3 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium +Kanamycin. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm. Day 2 : Prepare bacteria of Abs 600 of 0.1 in LB medium  Make a 1:10 dilution of each overnight culture in LB+Kanamycin (0.5mL ofculture into 4.5mL of LB+Kanamycin)  Measure Abs 600 of these 1:10 diluted cultures  Record the data in your notebook  Dilute the cultures further to a target Abs 600 of 0.1 in a final volume of 70 ml LB  medium + Kanamycin in 50 mL falcon tube. Prepare Copper ion solution in different concentration Generate 204.8 mM of Copper stock by dissolving 0.511g of copper salt (Copper (II) pentahydrate CuSO4 : 5H2O, MW: 249.69g/mol) in 10ml MilliQ and mix thoroughly  Then dilute the solution to 102.4, 51.2, 25.6, 12.8, 6.4, 3.2, 1.6 and 0.8mM with MilliQ And generate tubes of 10 different copper concentrations Prepare Copper ion solution in different concentration Generate 1280 mM of L-Arabinose stock by dissolving 1.922g of L-Arabinose, MW: 150.13g/mol) in 10ml MilliQ and mix thoroughly.  Then dilute the solution to, 640, 160, 40 and 10 mM with MilliQ  And generate tubes of 6 different copper concentrations Transfer copper ion solution and bacteria of Abs 600 of 0.1 in LB medium into 2ml deep-well plates: 20μl of each copper concentration in the order seen above in each row  1980μl of bacteria of Abs 600 of 0.1 in LB medium with antibiotic (ratio: 1μl of antibiotic per ml of LB) in each well  Use a blank deep-well plate with the same weight as the plate prepared 37°C, 200rpm  At 0，1， 2，3，4，5，6，7，8，9，10 h use a multi-tip pipette to transfer 200μl from each well into a plate reader compatible plate (COSTAR 96 plates)  Close the lid and transfer the plate into the plate reader  Adjust the temperature to and make a script with 2 protocols, one for OD and one for fluorescent measurements  OD protocol: 600nm  Fluorescence protocol: excitation filter 485±12nm, emission filter 520nm, bottom optic,  NOTE: The same protocol was used to measure protein expression in pBAD-RBS-sfGFP which require induction via L-arabinose and not copper. Arabinose concentrations used were the same. |


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# Team:BFSUICC-China/Experiments

**BFSUICC-China**

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[Parts](https://2018.igem.org/Team%3ABFSUICC-China/Parts) [BasicParts](https://2018.igem.org/Team%3ABFSUICC-China/Basic_Part) [CompositeParts](https://2018.igem.org/Team%3ABFSUICC-China/Composite_Part)

[Human Practices](https://2018.igem.org/Team%3ABFSUICC-China/Human_Practices)

# Test the function of parts and circuit

### PROTEIN EXPRESSION MEASUREMENTS

In order to show that our synthetic bacteria have a fully functional part that works, expression measurements were made. The construct was usually linked to superfold GFP. The fluorescent intensity can be measured in different ways and is directly related to the expression frequency of the construct (the more intense the signal the more expression you have). For most parts plate reader and microscopy experiments were done.

#### Plate Reading (Relative and Absolute Fluorescence)

> A plate reader is able to take optical density (OD600nm) and fluorescent measurements over time. OD is a measure of bacterial growth over time and fluorescence a measure of protein expression over time.

##### (I)Comparison between BBa\_K1555000 and BBa\_K2555000

> Plasmid: PSB4K5
>
> Chassis: E.coli Top 10

![](https://static.igem.org/mediawiki/2018/e/ec/T--BFSUICC-China--Factional_Test_1.png)

At 0，1，2，3，4，5，6，7，8，9 and 10h, fluorescent intensity and OD600 were measured
4-48 well plates

(II) BBa\_2555003 pBAD-RBS-sfGFP

![](https://static.igem.org/mediawiki/2018/d/d3/T--BFSUICC-China--Factional_Test_2.png)

At 0，1，2，3，4，5，6，7，8，9 and 10h measure fluorescent intensity and OD600 by plate reading

2 48-well plates

(III)BBa\_2555004 pCopA-RiboJ-RBS-sfGFP+pBAD-RBS-CueR

![](https://static.igem.org/mediawiki/2018/5/5f/T--BFSUICC-China--Factional_Test_3.png)

Bacteria with o, o.1, o.4, 1.6, 6.4 and 12.8 mM arabinose. At 0，5 and 10h fluorescent intensity and OD600 were measured.

2 48-well plates

（IV）For modeling and real world

BBa\_2555004 pCopA-RiboJ-RBS-sfGFP+pBAD-RBS-CueR(0.1mM Arabinose)

![](https://static.igem.org/mediawiki/2018/0/09/T--BFSUICC-China--Factional_Test_4.png)

Bacteria with o.1 mM arabinose. At 0，5 h fluorescent intensity and OD600 were measured.

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# Team:BFSUICC-China/Results

**BFSUICC-China**

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[Desgin](https://2018.igem.org/Team%3ABFSUICC-China/Design) [Experiments](https://2018.igem.org/Team%3ABFSUICC-China/Experiments) [Protocol](https://2018.igem.org/Team%3ABFSUICC-China/Notebook) [Interlab](https://2018.igem.org/Team%3ABFSUICC-China/InterLab) [Model](https://2018.igem.org/Team%3ABFSUICC-China/Model) [Results](https://2018.igem.org/Team%3ABFSUICC-China/Results) [Demonstrate](https://2018.igem.org/Team%3ABFSUICC-China/Demonstrate) [Improve](https://2018.igem.org/Team%3ABFSUICC-China/Improve) [Attributions](https://2018.igem.org/Team%3ABFSUICC-China/Attributions)

[Parts](https://2018.igem.org/Team%3ABFSUICC-China/Parts)

[Parts](https://2018.igem.org/Team%3ABFSUICC-China/Parts) [BasicParts](https://2018.igem.org/Team%3ABFSUICC-China/Basic_Part) [CompositeParts](https://2018.igem.org/Team%3ABFSUICC-China/Composite_Part)

[Human Practices](https://2018.igem.org/Team%3ABFSUICC-China/Human_Practices)

> > > > > > ###
> > > > > >
> > > > > > ### Construct Parts
> > >
> > > > #### Circuit
> > >
> > > > > ![](https://static.igem.org/mediawiki/2018/2/21/T--BFSUICC-China--resultnew1.jpg)
> > >
> > > > > We use the elcetrophoresis to detect whether the parts connect successfully.
> > > > > The right column is the marker and the strips on the left are the elcetrophoresis of BBa\_K 2555004. As shown in the diagram below, our parts has been ligated successfully. The process is achieved by digestion and ligation to pSB4K5 and pSB1C3.
> > > > >
> > > > > > #### Sensor Part ![](https://static.igem.org/mediawiki/2018/7/7a/T--BFSUICC-China--resultnew2.jpg)
> > > > >
> > > > > We change the backbone of BBa\_K2555000 in pUC 57 to pSB4K5 to pSB1C3. The right column is the marker, and the two strips on the left are restriction digestion products of BBa\_K2555000. The sizes of the two strips are both about 1128 bp.
> > > > >
> > > > > > #### Regulator Part ![](https://static.igem.org/mediawiki/2018/5/5e/T--BFSUICC-China--resultnew3.jpg) We change the backbone of BBa\_K2555002 in pUC 57 to pSB4K5 and pSB1C3. The right column is the marker, and the two strips on the left are registriction digestion products of BBa\_K2555002. The sizes of the two strips are both about 1819 bp. Test the function of parts
> > > > >
> > > > > #### Circuit Test the function of BBa\_K2555004 ![](https://static.igem.org/mediawiki/2018/0/09/T--BFSUICC-China--regulator1.png)

The results of a plate reader experiment with different arabinose concentration after five hours

> > ![](https://static.igem.org/mediawiki/2018/7/73/T--BFSUICC-China--regulator4.png)

Find the biggest dynamic range of arabinose
The results of dynamic range of BBa\_K2555004 with different arabinose concentrations after five hours.

##### Dynamic range

###### (highest value(concentration of copper ions is 2.048mM)-lowest value(concentration of copper ions is 0))/(lowest value(concentration of copper ions is 0)) Under o.1 mM arabinose BBa\_K2555004 has the biggest dynamic range, so the amount of CueR under o.1mM arabinose may has positive effect on activation of pCop A. Compare the function of BBa\_K15550000 and BBa\_K2555004 ![](https://static.igem.org/mediawiki/2018/f/fb/T--BFSUICC-China--regulator5.png) The results of a plate reader experiment with different copper and arabinose concentrations after five hours. Fluorescent increase rate=(output（high）-output(low))/(input(high)-input(low)) The relative fluorescence intensity, the increasing rate of relative fluorescence intensity and dynamic range of BBa\_K2555004 under 0.1 mM Arabinose were all significantly higher than that of BBa\_k1555000. Sensor part ![](https://static.igem.org/mediawiki/2018/4/47/T--BFSUICC-China--sensor1.png) Compare the function of BBa\_K25550000 and BBa\_K1555000 The results of a plate reader experiment with different copper concentrations after five hours. We optimized BBa\_k1555000 to be BBa\_K2555000. The relative fluorescence intensity of BBa\_K2555000 under different copper ion concentrations was higher than that of BBa\_k1555000, and the increasing rate of relative fluorescence intensity was higher than that of BBa\_k1555000. Regulator partTest the expression of CueR ![](https://static.igem.org/mediawiki/2018/b/b7/T--BFSUICC-China--regulator2.png) The results of a OD600 with different arabinose concentrations after five hours. With the increase of Arabinose concentration, the relative fluorescence value of the construct increased, which indirectly reflected the CueR protein expression of BBa\_K2555002 increased with the increase of Arabinose concentration. Test the effect of Cue R expression on growth of bacteria. ![](https://static.igem.org/mediawiki/2018/f/fd/T--BFSUICC-China--regulator3.png)