iGEM 2019 — Team Mines

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# Team:Mines/Notebook

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[Notebook](https://2019.igem.org/Team%3AMines/Notebook)
[Contribution](https://2019.igem.org/Team%3AMines/Contribution)
[Results](https://2019.igem.org/Team%3AMines/Results)
[Demonstrate](https://2019.igem.org/Team%3AMines/Demonstrate)
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HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3AMines/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AMines/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3AMines/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3AMines/Hardware)
[Measurement](https://2019.igem.org/Team%3AMines/Measurement)
[Model](https://2019.igem.org/Team%3AMines/Model)
[Plant](https://2019.igem.org/Team%3AMines/Plant)
[Software](https://2019.igem.org/Team%3AMines/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Mines)

## Mines Lab Notebook

Wet Lab General Summary

During the spring semester troubleshooting the sensing model using a well plate reader was the primary objective. The first half of summer was spent on ligating, and transforming the original binding sequence into PUC19 and E. Coli. The second half of the summer was devoted to redesigning a functional part to avoid the ligation problems primarily due to the small length of sequence. Much of the school year was devoted to manipulating the new part, and characterizing a Cadmium promoter.

Wet Lab Documentation

Day of 5/28/2019- Attempting Ligation of Binding Sequence into pMal

Digested pMAL-insertion vectors and pMAL (negative control) with btsa1 RE:

| Component | Volume(uL) |
| --- | --- |
| pMAL (38 ng/uL) | 5 |
| 10X CutSmart Buffer | 5 |
| Btsa1 resctriction enzyme | 1 |
| Nuclease Free Water | 39 |

Reaction Conditions: 55°C incubation for 20 minutes. No heat inactivation used (might gel purify in the
future, this test is just to verify the presence of insert)
Used a 10% agarose gel at 120V for 50 minutes:
1 - GeneRuler 1 kb plus
2 - Empty
3 - pMAL no digestion, no insert
4 - pMAL w/ insert, no digestion (2.2)
5 - pMAL digested, no insert
6 - pMAL w/ insert (1.1), digested
7 - pMAL w/ insert (2.1), digested

Day of 5/30/2019- Gel Digestion Analysis

Digested n-pMAL, 1.1-pMAL, 1.2-pMAL, and 2.1-pMAL with ApoI and BtaI
Used left over n-pMAL Btal digest
made concentrated digest (10ul insted of 50ul)

| Component | Volume (uL) |
| --- | --- |
| pMAL (38 ng/uL) OR pMAL -insert (unknown concentration) | 5 uL |
| 10X CutSmart Buffer | 1 uL |
| Btsa1, Apol, Restriction enzyme | 1 uL |
| Nuclease Free Water | 3 uL |

Ran Digest for 20 min, at 37°C for Apol and 55°C for Btal

inserted into gel, added 6 ul of loading dye to N-pMAL Btal digest, as there was around 40 ul of
solution to insert

Lane #

Ladder

Empty

n-pMAL #1 Btal digest

2.1-pMAL Btal digest

1.2-pMAL Btal digest

1.1-pMAL Btal digest

Empty

n-pMAL #1 Apo1 digest

2.1-pMAL ApoI digest

1.2-pMAL ApoI digest

1.1 -pMAL ApoI digest

![File:T--Mines--digestdig5-30.png](/wiki/images/thumb/d/d6/T--Mines--digestdig5-30.png/344px-T--Mines--digestdig5-30.png)
![File:T--Mines--digest5-30.jpeg](/wiki/images/thumb/d/d8/T--Mines--digest5-30.jpeg/800px-T--Mines--digest5-30.jpeg)

Day of 5/31/2019- TopoTA protocol

Goals:

Finish Plates---done
Run PCR on binding protin sequence---done
run Topo protocol---done
transform Topo protocal---done

| Component | Volume (uL) |
| --- | --- |
| NEB 2x MM (M0270s) | 25 |
| M13 FP | 1 |
| M13 RP | 1 |
| IDT Oligo | 1 |
| Nuclease-Free Water | 22 |

PCR Conditions:
1. 94 °C 5 min
2. 94 °C 0:30
3. 45 °C 0:30
4. 72 °C 0:20
5. Repeated 2-4 30 cycles

5. 72 °C 7:00
6. 4 °C hold (was about 2 hours on hold before TOPO TA

Ran gel of PCR:

2% agarose gel, 110V, 40 minutes with 100 base Generule Ladder
Added 2 uL loading dye and 10 uL PCR product, loaded 10 uL of ladder and samples

Added 2 Lanes:
Generuler 100 bp ladder
---
PCR Replicate 1
---
PCR Replicate 2

PCR gel 3\_31\_2019.jpg
TOPO TA CLoning of bnd PCR:
Followed TOPO TA Cloning kit protocol by Thermo Fisher

![File:T--Mines--digest5-31.jpeg](/wiki/images/thumb/4/4a/T--Mines--digest5-31.jpeg/450px-T--Mines--digest5-31.jpeg)

|  | Volume (uL) |
| --- | --- |
| PCR product | 4 |
| Salt solution | 1 |
| TOPO Vector | 1 |

Transformation
DH5-alpha
Followed TOPO TA Cloning protocol for One Shot DH5-alpha for transformation by Thermo Fisher
Did not use X-gaL solution on our plates
Tempature of water bath was not accurate, ranged from 40-45°C at 30 seconds
Added 900 uL of SOC to each solution instead of 250uL
added 100 uL of solution to plates with two controls for each sample
We have produced 6 plates and have 300 uL of solution left over for each sample
Plates are labeled 2.1 and 2.2, with the first number corrisponding to the sample number and the second
to plate number.. The controls are simply labled 2 for sample number

Made LB/ amp plates:

Added 20.0 grams agar to 1 L LB
Autoclaved on liquid setting (cover with foil and autoclave tape, loosen cap a bit so it doesn't explode
when applicable)
Let cool down to touchable temperature (when you can comfortably touch the flask for 10 seeconds)
Added 1 mL of 1000X ampicillin
Poured into plates and let dry. The lid was left slightly off to prevent condensation until it
cooled
Stored in fridge with lids on the bottom

Possible Errors in Cloning/Transformation
We never timed the mixture of the TOPO solution, we left it to run for more like ten minuets instead of
the perscribed 5. This could have lead to a degridation of the TOPO product
One shot cells were left on ice for a long time ~40 min, an not submerged, meaning they were posible too
warm to undergo heat shock
Heat shock was not an accurate tempature
we added 6 uL of TOPO product instead of 4, I cant think of a way that this would effect the
results.

Day of 6/04/2019- Incubation of Colony

Goals
Incubate lone colony from plate 1---done
restreak plates with left over solution---done
design plasmid for IDT to create

Icubate Colony
colony was removed from plate and placed in 5 mL of LB amp solution and left to incubate in shaker
overnight
Streak Plates
Added 300 ml of transformed bacteria to plates instead of 100, solution drained to cover of plates after
they were fliped- left overnight

Results from 5/31
The controls had growth but the LB amp plates exibited no growth exept for a lone colony. This colony is
most likely a mutation or forgen bacteria that managed to make it into the plate

Day of 6/05/2019- Analysis of Growth

Goals
make IDT vector
analyze lone bacteria colony that was growing

Analyze
Took the absorbance of the incubated ecoli solution- 1.287 at 600 nm, indicating growth
The plates showed no growth, indicating that the lone bacteria colony is most likely extranious.
To preserve the colony, the solution was replated and then diluted to two LB amp plates and two LB amp
solutions
The left over solution was then run through a miniprep to extract the plasmid from it and then run a
gell on the plasmid to see if it has the insert (ZymoPure Plasmid miniprep)

Plates produced a matt of colonies

Day of 6/06/2019- New PCR Product

Goals
Make IDT vector
Analize plates---done
rerun TOPO reaction on New PCR product----done

Analize
plates produced matts
PCR
We PCRed two products, the IDT Binding Vector OLIGO in tubes 1 and 2

|  | Volume (uL) |
| --- | --- |
| Topo TA PCR product | 25 |
| Cut Smart | 5 |
| EcoRI | 1 |
| Denucleased water | 19 |
| Total | 50 |

| Component | Volume (uL) |
| --- | --- |
| NEB 2x MM (M0270s) | 25 |
| M13 FP | 1 |
| M13 RP | 1 |
| IDT Oligo | 1 |
| Nuclease-Free Water | 22 |

PCR steps were taken from MEB for the Taq 2 MM
95 at 1 min
30 cycles
95 at 30 sec
45 at 60 sec
68 at 20 sec
68 at 5 min
4 deg hold
All of 1-TOPO TA Mini 2115-a was used in the PCR the OLIGO and tube 2 were placed in box 2

| Component | Volume (uL) |
| --- | --- |
| NEB 2x MM (M0270s) | 25 |
| M13 FP | 1 |
| M13 RP | 1 |
| TOPO TA Mini | 22 |
| Nuclease-Free Water | 1 |

A 1.5 mL tube was prepared by the table above. Then placed in a 37 °C hot water bath for 30 minutes.
Blue White screening of the Lone Colony
Using 20 mg/ml of X-gal in DMSO, we plated one of the colonys on a LB/Amp plate
we heated the plate to 37°C for 30 min, we added 40 ul of x-gal soution to plate and incubated
......
We added 50 ul of Last Hope second gen to the plate in order to get more differentiation, and incubated
overnight at 37°C

|  | Volume (uL) |
| --- | --- |
| PCR product | 4 |
| Salt solution | 1 |
| TOPO Vector | 1 |

Followed TOPO TA Cloning kit protocol by Thermo Fisher
left mixer on for 7 min
DH5-alpha
Followed TOPO TA Cloning protocol for One Shot DH5-alpha for transformation by Thermo Fisher
used X-gaL solution on 1 plate
3 plates were produced, 1 control, one with x gal, and one without

Gel Ran
lane 1 100 bp ladder
lane 3 new pcr product of TopoTA Cloning
lane 4 old pcr made

last hope minipreped and pcred.jpg

visibility was low, to bars of similar length were present, gel was stored in fridge for furthure
analysis.

![File:T--Mines--digestlasthope6-6.jpeg](/wiki/images/thumb/d/d3/T--Mines--digestlasthope6-6.jpeg/800px-T--Mines--digestlasthope6-6.jpeg)

Day of 6/07/2019- Creation of Freezer Stock

Goals
Check results on new topo reaction---done
create freezer stocks---done
Created freezer stocks out of the Last Hope 2nd Gen

2ND TOPO Rxn

IMG\_20190607\_143407390.jpg
10 white colonys and 3 blue colonys were taken from this plate and inserted into 5 ml LB solutions to
incubate
these colonys are called A New Hope #1-10 and A New Hope Blue #1-3

![File:T--Mines--plate6-07.jpeg](/wiki/images/thumb/e/e3/T--Mines--plate6-07.jpeg/450px-T--Mines--plate6-07.jpeg)

Day of 6/10/2019 White Blue Colony Screening

Goals
Check A New Hope Colonys for insert
make more pMAL stock
Mini Prep of 4 white colonies and 2 blue (negative) colonies (New Hope batch)
1 ml of baterial culture was added to 1.5 ml tubes. Then the standard mini prep protocol was preformed.
Mini preps of White colonies 1-4, and Blue
colonies 1-2 were made.
pMAL Stock
pMAL plasmid was taken out and put into 5ml LB to grow

The 2nd TOPO Rxn B/W plate turned almost completely blue
Before:
2nd TOPO Rxn B\_W plate.jpg
After
2nd Topo Rxn B\_W plate next day.jpg
Mini Gel of Colonies Ran
Lanes
Gener Ruler 1 KB Ladder 10ul
---------
White 1 15 ul
White 2 15 ul
White 3 15 ul
White 4 15 ul
Blue 1 15 ul
Blue 2 15 ul
Conditions: 90 Volts 90 minutes

![File:T--Mines--plate6-10.jpeg](/wiki/images/thumb/e/e0/T--Mines--plate6-10.jpeg/800px-T--Mines--plate6-10.jpeg)
![File:T--Mines--2plate6-10.jpeg](/wiki/images/thumb/2/21/T--Mines--2plate6-10.jpeg/450px-T--Mines--2plate6-10.jpeg)

Day of 6/11/2019 MiniPrep of Colonies

Goals
Insert Binding Portion into pMAL proten---in progress
test A New Hope minipreps for insert--- in progress

Topo To pMAL
Took pMAL precursor LB solution, put 100 uL on prewarmed LB/Amp plate, incubated at 37 for a day.

Minipreps
Made master mix

|  | Volume (uL) |
| --- | --- |
| PCR product | 4 |
| Salt solution | 1 |
| TOPO Vector | 1 |

5 ul of master mix was pipeted into 6 centrifuge tubes, and 6 samples of minipreped DNA was inserted
into a seperate tube 5 ul each
samples were submerged in 37 degree water overnight

Gel Run of Digest
Lanes
100 Base Pair Ladder
2.
3. B1
4. B2 (Mostly Empty)
5. W1
6. W2
7. W3
8. W4
120 Volts for 45 minutes

Day of 6/12/2019 Gel Analysis of Colonies

Goals
Run gel of digested minipreps---done

Gel
standard load
very odd results
gel was soaked in ETBR soulution overnight

![File:T--Mines--digest6-12.jpeg](/wiki/images/thumb/6/6c/T--Mines--digest6-12.jpeg/800px-T--Mines--digest6-12.jpeg)
![File:T--Mines--digestdig6-11.png](/wiki/images/thumb/5/59/T--Mines--digestdig6-11.png/275px-T--Mines--digestdig6-11.png)

Day of 6/13/2019- PCR analysis

Goals
fugure out what the gel resutls mean
fix IDT Vector---done
Run Topo TA try 2 PCR products through gel---done
Gel
Possibly some combination of superfoiled, uncoiled, and cut
IDT vector
changes recorded in notes on vector
PCR
Standard loading conditions, no results

Day of 6/14/2019- Gel Analysis of Second TopoTA

Goals
Run a gel of The Last Hope, The PCR that was inserted, and All the PCR products produced for TOPO TA try
2

Gel
Added 20 uL of solution to each lane, with an additional 5 ul of loading dye, added 25 ul of minipreped
and digested last hope-2118

Lane 1:Gene ruler 1kb
Lane 2: 100 bp ladder
Lane 3:
Lane 4: 2114-a
Lane 5: 2114-b
lane 6: 2118
Lane 7: Topo rxn 2, most likely used for TOPO insert, PCR
Lane 8: TOPO rxn PCR #2

All results are positive, even if they are faint. most concerning is the 2118 missing its larger back
bone, not even showing up in the gel. the lines also are closer to the 50 bp line than the 100 bp line,
when the insert should be about 120 bp

\*\*\*\*\*CORRECTION (6/7/19)\*\*\*\*\*
The ladder used in Lane 2 is the NEB Quickload 100 bp ladder (N0467S). The image of the ladder is below.
The bands are just above the 100 bp mark at the very bottom of the ladder, indicating that the DNA cut
from TOPO vector was successfully inserted. The error is likely due to referencing the Fisher 100 bp
ladder instead of the NEB quickload 100 bp ladder.

![](https://static.igem.org/mediawiki/2019/a/a0/T--Mines--ladder6-14.png)
![](https://static.igem.org/mediawiki/2019/thumb/c/c8/T--Mines--digest6-14.jpeg/320px-T--Mines--digest6-14.jpeg)
![](https://static.igem.org/mediawiki/2019/3/30/T--Mines--digestclose6-14.jpeg)

Day of 6/18/2019 Miniprep of pMal precursor

Goals
take swipe from pMAL to put in lb solution---no
pcr amplify sections of topo try 2 to check for insert---no
check gel for change---done
miniprep a last hope some of the pMAL precursor solution to prepare for insert---done
PCR amplify

m13 primers are missing: cannot continue

| Component | Volume (uL) |
| --- | --- |
| NEB 2x MM (M0270s) | 25 |
| M13 FP | 1 |
| M13 RP | 1 |
| TOPO TA Mini #2 | 22 |
| Nuclease-Free Water | 1 |

LB solution
not done

check gel
the stains have spread, lowering the resolution

miniprep
followed zymo protocal
produced two solutions: pmal and last

Day of 7/10/2019- Freezer Stock Recreation

Individual colonies were selected from each plate and suspended in 5 ml of LB/Amp solution. Then
incubated at 37 C at 230 RPM. Two colonies from Pmal 1 were incubated.
6 freezer stocks were created using 600 ul of incubation solution, and 400 ul of 50% glycerol.

| Last Hope 1 | Growth on plate | Incubated over night | Successful incubation |
| --- | --- | --- | --- |
| Last Hope 2 | Growth on plate | Incubated over night | Successful incubation |
| Sensing 1 | Growth on plate | Incubated over night | Successful incubation |
| Sensing 2 | Growth on plate | Incubated over night | Successful incubation |
| Pmal 1 | Growth on plate | Incubated over night | Successful incubation |
| Pmal 2 | No growth |  |  |
| igem GFP | No growth |  |  |
| igem GFP | No growth |  |  |

Day of 7/23/2019- Transformation with Control

The competent cells and ligation mixture were prepared, and a control of puc19 at 10 pg/ul was
also made with 5 ul of DNA added.
Competent cells were thawed on ice.
the cells were mixed with the plasmid as above.
The mixture was placed on ice for 10 minutes.
the cells were heat shocked for 30 seconds and placed back on ice.
950 ul of S.O.C. was added to the the solution
Solution was incubated at 37 C for 90 minutes shaking at 250 rpm.
Ampicillin selection plates were warmed
Plates of 10 and 100 ul of cells were spread.
Plates were incubated overnight at 37 C over night.

| Component | Volume |
| --- | --- |
| Chemically Competent cells | 50 ul |
| IDT Binding Plasmid (5ng/ul | 1 ul |

Day of 9/29/2019 PCR of New Binding Sequence

Goals:

PCR IDT Gene Block

Prmers with and without tag were synthesized from IDT

PCR with no tag primers preformed

PRC Settings:

30 ||| 15-60 seconds | 55C | 30 |
| --- | --- | --- |
| 1 minute/kb | 68°C | 1:00 |
| final extention | 68C | 5:00 |
| hold | 4C |  |
| Final Extension | 68°C | 5 minutes |
| Hold | 4-10°C |  |

Day of 10/09/2019 PCR Product Analysis

Ran gell of pcr product of dna 2133

nanodrop concentration 325 ng/ul

Gel:

Lane 1: 100 bp Ladder (6 ul)

Lane 2: empty

Lane 3: 5 ul DNA 1 ml loading Dye

Results:

![File:T--Mines--Gel10172019.jpeg](/wiki/images/thumb/5/56/T--Mines--Gel10172019.jpeg/337px-T--Mines--Gel10172019.jpeg)

Day of 10/15/2019 Hifi Assembly and Plate Preparation

Hifi Assembly of IDT dna into Igem vector

Dna 2133 was diluted in a 1:1 ratio with dn water

4 ul of digested backbone was placed in pcr tube

added 1 ul of diluted dna 2133

5 ul of dn water added

10 ul of hifi master mix added

POSITIVE CONTROL

5 ul of puc 19 control added

5 ul of NEBuilder Positive control added

10 ul of hifi master mix

both samples put in pcr maching at 50 deg. C for 15 min

created chloramphencol plates

Protocol:

Mix:

10 g of Agar

12.5 g of Broth

500 mL of Deionized water

Autoclave

Add 12.5 mg of choramphencol after cooled to touch

Approximately 20 mL of solution was added to each plate. Plates were left for 30 minutes to solidify.

21 plates prepared 4 of normal lb for control purposes and the rest were chloramphenocol plates.

Day of 10/16/2019 Transformation of RFP gene and Binding gene

Goals

transfom and plate both positive control and DNA 2213 in ecoli

Rehydrolise and transform cad sensing protine for characterisation

Transform

nanodroped postive control and DNA 2213 (now DNA 2136)

Postive control- 1381 ng/ul

DNA 2136-1580 ng/ul

did math instead- got 18.1 ng/ul

need 1 pg -100 ng

5 ul of each sample required for transformation- assume postive control is similar

Rehydrolise

put 10 ul of nulcease free water to get 10 mg/ul

placed 1 ul of Cad protine for transformation

Transformation proctocol

unthawed three chemicaly compitent cells

c for positive control

b for DNA 2136

2h for cadA sensing

skiped dilution step, went straight to plating 10 ul and 100ul dillutions


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# Team:Mines/Experiments

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TEAM

[Team Members](https://2019.igem.org/Team%3AMines/Team)
[Collaborations](https://2019.igem.org/Team%3AMines/Collaborations)

PROJECT

[Description](https://2019.igem.org/Team%3AMines/Description)
[Design](https://2019.igem.org/Team%3AMines/Design)
[Experiments](https://2019.igem.org/Team%3AMines/Experiments)
[Notebook](https://2019.igem.org/Team%3AMines/Notebook)
[Contribution](https://2019.igem.org/Team%3AMines/Contribution)
[Results](https://2019.igem.org/Team%3AMines/Results)
[Demonstrate](https://2019.igem.org/Team%3AMines/Demonstrate)
[Improve](https://2019.igem.org/Team%3AMines/Improve)
[Attributions](https://2019.igem.org/Team%3AMines/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AMines/Parts)
[Basic Parts](https://2019.igem.org/Team%3AMines/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AMines/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3AMines/Part_Collection)

[SAFETY](https://2019.igem.org/Team%3AMines/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3AMines/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AMines/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3AMines/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3AMines/Hardware)
[Measurement](https://2019.igem.org/Team%3AMines/Measurement)
[Model](https://2019.igem.org/Team%3AMines/Model)
[Plant](https://2019.igem.org/Team%3AMines/Plant)
[Software](https://2019.igem.org/Team%3AMines/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Mines)

![](https://static.igem.org/mediawiki/2018/a/a4/T--CO_Mines--mines_title_3_2.jpeg)
![](https://static.igem.org/mediawiki/2018/1/11/T--CO_Mines--mines_spacer.jpeg)

# Miniprep Protocols

[Zyppy Plasmid Miniprep Kit from
*Zymo Research*](https://static.igem.org/mediawiki/2018/1/11/T--CO_Mines--miniprep_protocol.pdf)

[Monarch Plasmid
Miniprep Kit from *New England BioLabs*](https://www.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010)

All centrifugation steps should be carried out at 16,000 x g (~13,000 RPM).

Store Plasmid Neutralization Buffer at 4°C after opening, as it contains RNase A.

1. Pellet 1.5 ml bacterial culture by centrifugation for 30 seconds. Discard supernatant.
2. Resuspend pellet in 200 μl Plasmid Resuspension Buffer. Vortex or pipet to ensure cells are completely
   resuspended. There should be no visible clumps.
3. Lyse cells by adding 200 μl Plasmid Lysis Buffer. Invert tube immediately and gently 5–6 times until color
   changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute.
4. Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer. Gently invert tube until the color is
   uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.
5. Clarify the lysate by spinning for 2 minutes.
6. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer. Carefully transfer supernatant to
   the spin column and centrifuge for 1 minute. Discard flow-through. Centrifuge for 1 minute.
7. Add 400 μl of Plasmid Wash Buffer and centrifuge for 1 minute.
8. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come
   into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute before inserting it
   into the clean microfuge tube.
9. Add ≥ 30 μl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute
   DNA.

# Making Lysogeny Broth (LB) medium

**Liquid LB (for 1 L)**

**Ingredients:**

* 10 g Tryptone
* 5 g Yeast Extract
* 10 g NaCl

**Steps:**

1. Weigh ingredients out into an Erlenmeyer flask.
2. Add DI water to 1 mL and swirl flask to mix.
3. Cover the top of the flask with aluminum foil and autoclave tape.
4. Autoclave for 20 minutes with a slow exhaust.
5. Allow LB to cool to 55 °C before adding any antibiotics.

**LB-agar plates**

Follow the same steps as above, but add 15 g of Agar before adding DI water.

Once any necessary antibiotics have been added, pour a thin layer of LB-agar into Petri dishes. If any
bubbles form, they can be removed by holding a flame over them.

[LB Agar
Plates Protocols for Bacteria from *Addgene*](https://static.igem.org/mediawiki/2018/8/89/T--CO_Mines--LB_agar_protocol_addgene.pdf)

# Restriction Digestion

1. Select restriction enzymes to digest your plasmid.
2. Determine an appropriate reaction buffer by reading the instructions for your enzyme.
3. In a 1.5mL tube combine the following:

* DNA (~1 µL)
* Restriction Enzyme(s)
* Buffer
* BSA (if recommended by manufacturer)
* nuclease free H2O (nfH2O) up to total volume
A typical restriction digestion reaction could look like this:

+ 1 µg DNA
+ 1 µL of each Restriction Enzyme
+ 3 µL 10x Buffer
+ 3 µL 10x BSA (if recommended)
+ x µL nfH2O (to bring total volume to 30-50 µL)

4. Mix gently by pipetting.
5. Incubate tube at the manufacturer described temperature and time.
6. Use gel electrophoresis to confirm if the digestion was successful.

# T4 Ligation Protocol

The reaction is performed on ice. The reaction is scaled to the specifications of the Thermo
Fisher T4 DNA Ligase Instructions. The ratio of Insert to Vector was Modified for some
ligations.

1. In a 1.5mL tube combine the following:

* Vector and Insert DNA
* T4 Ligase
* Buffer
* nuclease free H2O (nfH2O) up to total volume
A typical ligation reaction could look like this:

+ 50 ng Vector DNA
+ 37.5 ng Insert DNA
+ 2 µL
+ 2 µL 10x T4 DNA Ligation Buffer
+ x µL nfH2O (to bring total volume to 20 µL)

2. Mix gently by pipetting.
3. Incubate tube at the 16°C overnight or at room temperature for 2 hours.
4. Use gel electrophoresis to confirm if the ligation was successful.

# PCR reaction

[PCR Protocols for Taq
DNA Polymerase from *New England BioLabs*](https://static.igem.org/mediawiki/2018/a/ae/T--CO_Mines--PCR_protocol.pdf)

**For a 25 µL reaction:**

* 2.5 µL of 10x Taq master mix
* 0.5 µL of 10 mM dNTPs
* 0.5 µL of 10 uM forward primer
* 0.5 µL of 10 uM reverse primer
* 1 µg of template DNA
* Top to 25 µL with nuclease free water

**Thermocycling conditions for a routine PCR:**

* Denaturing: 95 °C for 30 seconds
* Cycle: Denature for 30 seconds at 95 °C, Anneal for 30 seconds at temperature, Extend for 1
  min/kb at 68 °C
* Hold at 4 °C

# Gel electrophoresis

**(For a 1 kb gel)**

1. Measure out 0.5 g of agarose and add to an Erlenmeyer flask.
2. Measure out 50 mL of 1x TAE buffer and add to the flask.
3. Swirl to mix agarose into the TAE and microwave until agarose is fully dissolved, checking to
   ensure the mixture does not boil over.
4. Add 2 µl of ethidium bromide and swirl to mix.
5. Pour mixture into a gel tray and add gel comb. Allow the gel to sit until solid, about 30
   minutes.
6. Once the gel is solidified, remove the comb and move tray into electrophoresis unit and add 1X
   TAE until electrophoresis unit is filled.
7. Add 1 µL of loading dye to 5 µL of DNA.
8. Add 6 µL of DNA ladder to first gel lane and 6 µL of each labeled sample DNA to the rest of the
   lanes.
9. Plug the electrophoresis unit in and run at 120 V for around 40 minutes.
10. Turn the power off and view gel under ultraviolet light.

# DNA Transformation into *E.coli*

[Bacterial
Transformation Protocols from *Addgene*](https://static.igem.org/mediawiki/2018/f/fd/T--CO_Mines--transformation_protocol.pdf)

1. Allow competent cells to thaw on ice and take out selecting plates to warm up.
2. Add 2 µL of DNA to 25 µL of competent cells.
3. Incubate the competent cells on ice for 30 minutes.
4. Heat shock the cells by incubating them at 42 °C for 30 seconds and then immediately return them
   to ice for 2 minutes.
5. Add 500 µL of LB to the cells and place in shaking incubator at 250 rpm and 37 °C for 1 hour.
   Place plates into a 37 °C incubator to warm up.
6. Take a sterile loop and plate cells, then return the plates to the incubator overnight.
7. Check the plates for colonies after incubation.

# TOPO TA Cloning

[TOPO PCR Protocols
from *Thermo Fisher Scientific*](https://static.igem.org/mediawiki/2018/c/c1/T--CO_Mines--TOPO_protocol.pdf)

1. Add 1 µL of PCR DNA with 1 µL of Topo cloning vector.
2. Incubate the mixture at room temperature for 5 minutes.
3. Perform a transformation of competent cells using the Topo mixture and plate.

# Kinetic Scan Procedure

1. All colonies are grown overnight in 5 mL of LB and the respective antibiotic.
2. The next day a 1/10th dilution is performed and then the OD600 is measured and used to make
   seperate 0.1 OD600 colonies.
3. The 0.1 colonies are incubated at 37°C for 1 hour.
4. The OD600 is measured.
5. The well plate is set up by placing 100 uL of growth media into each well being used. A set of
   serial dilutions is then performed using growth media with Cd+2 to achieve the
   desired Cd+2 concentration.
6. 100 uL of the colonies are placed into each respective well plate.
7. The 96 well plate reader is then set to a kinetic scan for 5 hours reading both OD600 and
   fluorescence. The fluorescence reading is set to 490 excitation and 510 emission. The rotation
   is set to 275 cpm and the temperature to 37°C.

# HiFi Assembly Protocol

Based on [HiFi Protocol from New England BioLabs](https://www.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol)

|  |  |  |  |
| --- | --- | --- | --- |
|  | **Recommended Amount of Fragments Used for Assembly** | | |
| 2–3 Fragment Assembly\* | 4–6 Fragment Assembly\*\* | Positive Control✝ |
| Recommended DNA Molar Ratio | vector:insert = 1:2 | vector:insert = 1:1 |  |
| Total Amount of Fragments | 0.03–0.2 pmols\*  X μl | 0.2–0.5 pmols\*\*  X μl | 10 μl |
| NEBuilder  HiFi DNA Assembly Master Mix | 10 μl |
| Deionized H2O | 10-X μl | 0 |
| Total Volume | 20 μl |

Prepare the above mixture on ice in a PCR tube. Then incubate at 50 °C for 15 minutes in the Thermocycler. The products can be used in a standard transformation protocol.

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# Binding:

We
found that the sequence is too small to tell if it is present in the ligation
with a pMAL vector. When trying to isolate the segment (which is about 75 bp
long), the concentration of DNA was too small to be see in the gel. This can be
seen in Figure 1.

![](https://static.igem.org/mediawiki/2018/5/5f/T--CO_Mines--pUC19Gel.jpeg)

**Fig. 1-**  Digestion of the pUC19 vector
with the binding insert with a 100 bp.

Based on the gels above, we saw that the segment was too small in the gel. We tried to use a different restriction endonuclease (BsaI) which would result in larger fragments. The expected gels from this digestion with and without the segment are shown in Figure 2.

![](https://static.igem.org/mediawiki/2018/1/1d/T--CO_Mines--expectedGels.jpeg)

**Fig. 2 -**  Expected gels of size of fragments by cutting (a) the pMAL-p4x with the cadmium binding peptide (CBP) sequence and (b) the empty pMAL-p4x vector. The image was produced by the NEBCutter tool [1].

When running the gels, some of the ligations were negatives (wells a.4, b.5-b.7); however, the other wells were inconclusive as can be seen in Figure 3. We believe this was due to some interference in the digestion or that the insert was too short to be able to tell easily through restriction endonuclease digestion. The results could also be inconclusive due to bad samples as evident by the smearing.

![](https://static.igem.org/mediawiki/2018/c/cc/T--CO_Mines--BsaIDigestion.jpeg)

**Fig. 3 -**  Digestion of binding ligation to determine if the peptide insert is present

To find out the inconclusive results, we plan on replicating the ligation and digestion to see if there was interference in the procedure. We also plan to sequence the ligation to determine if the insert is in the vector without using restriction endonucleases.

# Composite Binding Synthesis

We synthesized a new [composite binding sequence](http://parts.igem.org/Part%3ABBa_K3297000) that contains a tac promoter with the maltose binding protein and the binding sequence down stream. This sequence should translate
the moltose binding protien with the binding sequence hanging off by a small linker. The purpose of the Maltose binding protein is to transport the binding protein to the periplasmic space in order to increase binding efficiency and lower the risk to the cell, and to stabilize the smaller protein. Primers for the sequence were made into to run PCR.
Gel of PCR:

![File:T--Mines--Gel10172019.jpeg](/wiki/images/thumb/5/56/T--Mines--Gel10172019.jpeg/337px-T--Mines--Gel10172019.jpeg)

While the gel indicated some fragmentation, the concentration was found to be 325 ng/ml. This product and the pSB1C3 linearized plasmid were combined using
Hifi Assembly. Subsequent transformation into chemical competent E. Coli resulted in no collony growth. This is was likely due to a storage error with the linearized plasmid.

- Test the peptide to find out the selectivity and properties
- Use noncanonical amino acids with more thiol and other nucleophilic groups to improve the binding affinity and lower the peptide toxicity

# Sensing:

The first test of the sensing plasmid from IDT did not show any difference in expression of the GFP between different concentrations of cadmium. The amount of fluorescence when adjusted to the amount of cells decreased with time. This could be explained due to the cells not being viable. The cells had a doubling time of about 9 hours which significantly longer than what is typical for E. coli. The slow growth may be due to the temperature of the well-plate reader being room temperature instead of 37°C. The cells were from an old plate possibly reducing the cell viability. Another possibility for the stunted growth is that the well on the plate did not provide optimal conditions for the cells. Some of the results from this test can be seen in Figure 4 and 5.

![](https://static.igem.org/mediawiki/2018/7/73/T--CO_Mines--FlourescenceGraph.jpeg)

**Fig. 4 -** Fluorescence per cell for the first sensing test. The error bars represent 90% confidence.

![](https://static.igem.org/mediawiki/2018/3/33/T--CO_Mines--AbsGraph.jpeg)

**Fig. 5 -**  Growth of cells for first sensing test. The error bars represent a 90% confidence interval

Some explanations of there being no difference in expression of the GFP between the different concentrations of cadmium are as followed:

* There is a nonspecific promoter ahead of the segment without a terminator.
* There is cadmium present in the media due to the media being in a glass container.
* There is promoter leakage from within the plasmid.

To eliminate these possible sources two routes were taken. The promoter, regulatory gene, an GFP sequence was attempted to be moved to the iGEM pSB1C3 backbone. The pSB1C3 backbone has 2 terminators before the section where the sequence would be added, However, the transformation was not successful.

The other route was using the IDT vector again, but using cells from a new plate, using media that was filter sterilized in plastic, and setting the plate reader to 37°C. These steps would make it so the media has minimal or no heavy metal contamination, that the new colonies were more likely to be viable, and use the optimal growth conditions. The results of the second test are shown in Figure 6. They show that all the colonies grew with about the same doubling time which was around 70 minutes. This is a higher value than the expected for  *E. coli*, but does show viable growth.

![](https://static.igem.org/mediawiki/2018/2/2c/T--CO_Mines--doublingTimeGraph.jpeg)

**Fig. 6 -**  doubling time for different cadmium concentrations for the second sensing test. The error bars represent a 90% confidence interval

This second test had an overflow of the fluorescence which may be because the emission and excitation wavelengths were too close to each other. Another explanation of the overflow reading that the GFP concentration was higher than expected due to more expression by the cells.

![](/wiki/images/thumb/4/4c/T--Mines--Floursencespring2019.png/800px-T--Mines--Floursencespring2019.png)

**Fig. 7 -**  Fluorescence over time

The test was run again with the same concentrations and the results show the sensing was acting the same as the negative control. OD analysis of the cells indicates the doubling time of the cells was approximately 90 minutes. This can be seen in  **Fig. 8** The results indicate that the plasma may have been lost during storage.

![File:T--Mines--Doubling time regression spring 2019.png](/wiki/images/f/f8/T--Mines--Doubling_time_regression_spring_2019.png)

**Fig. 8-** Doubling Time regression

###

In the future we plan to:

* Move the insert into the iGEM plasmid
* Test sensing fluorescence and sensitivity

# Further steps:

Beyond the short term goals, we are planning on longer term goals to improve and combine the different components. We plan to:
Use noncanonical amino acids to improve the affinity of the binding peptide with cadmium
Increase the cadmium resistance to the sensing plasmid.
Add the cadC promoter and regulatory gene in front of the binding protein fusion to selectively make the protein.
In the future the new component part can replace the tac promoter with a cadmium specific promoter to increase the specificity and reduce strain on the bacteria when no cadmium is present in the environment.

## Reference

**[1]**  Tamas Vincze, Janos Posfai, Richard J. Roberts; NEBcutter: a program to cleave DNA with restriction enzymes,  *Nucleic Acids Research* , Volume 31, Issue 13, 1 July 2003, Pages 3688–3691, https://doi.org/10.1093/nar/gkg526