iGEM 2021 — Team Missouri_Miners

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# Team:Missouri Miners/Notebook

## Lab Notebook

## 10/23/2020 - Transfromation

#### Start Time

4:00, 10/23/2020

#### Purpose

Transformation of a variety of different parts, including RFP, GFP, YFP, and a protein coding sequence. Debugging the transformation protocol is of the utmost importance, as getting high enough concentrations for restriction digest can not be completed without it. In the past the Missouri S&T iGEM team has struggled with transforming out of the kitplates. Until this is resolved the team cannot maintain the stock of DNA in high enough concentrations to make any meaningful progress.

#### Protocol

ig Sa Chemically Competent Cells Protocol (90 minute protocol)

* 4 x 50 uL chemically competent cells
* 1 ul of each plasmid DNA
* 1 ul of each plasmid DNA

#### Products

##### Transformation Epi Tubes

| Sample Label | Source Sample Label | Description |
| --- | --- | --- |
| 10/23/2020 CT1 | RFP BBa\_J04450 Control DNA | 2019 KP5, Well 23O |
| 10/23/2020 CT2 | BFP BBa\_K592009 | 2019 KP1, Well 19E |
| 10/23/2020 CT3 | YFP BBa\_K1033910 | 2019 KP3, Well 6L |
| 10/23/2020 CT4 | BBa\_K118000 | 2019 KP6, Well 18D |

##### Petri Dishes

| Sample Label | Source Sample Label | Description |
| --- | --- | --- |
| 10/23/2020 CT1 1 50ul | 10/23/2020 CT1 | RFP BBa\_J04450 Control DNA on 9/3/2020 LB Agar (C) Plates |
| 10/23/2020 CT1 2 100ul | 10/23/2020 CT1 | RFP BBa\_J04450 Control DNA on 9/3/2020 LB Agar (C) Plates |
| 10/23/2020 CT1 3 100ul | 10/23/2020 CT1 | RFP BBa\_J04450 Control DNA on 8/25/2020 LB Agar Plates |
| 10/23/2020 CT2 1 50ul | 10/23/2020 CT2 | BFP BBa\_K592009 on 9/3/2020 LB Agar (C) Plates |
| 10/23/2020 CT2 2 100ul | 10/23/2020 CT2 | BFP BBa\_K592009 on 9/3/2020 LB Agar (C) Plates |
| 10/23/2020 CT2 3 100ul | 10/23/2020 CT2 | BFP BBa\_K592009 on 8/25/2020 LB Agar Plates |
| 10/23/2020 CT3 1 50ul | 10/23/2020 CT3 | YFP BBa\_K1033910 on 9/3/2020 LB Agar (C) Plates |
| 10/23/2020 CT3 2 100ul | 10/23/2020 CT3 | YFP BBa\_K1033910 on 9/3/2020 LB Agar (C) Plates |
| 10/23/2020 CT3 3 100ul | 10/23/2020 CT3 | YFP BBa\_K1033910 on 8/25/2020 LB Agar Plates |
| 10/23/2020 CT4 1 50ul | 10/23/2020 CT4 | BBa\_K118000 on 9/3/2020 LB Agar (C) Plates |
| 10/23/2020 CT4 2 100ul | 10/23/2020 CT4 | BBa\_K118000 on 9/3/2020 LB Agar (C) Plates |
| 10/23/2020 CT4 3 100ul | 10/23/2020 CT4 | BBa\_K118000 on 8/25/2020 LB Agar Plates |

#### Results

Barren selection plates, low transformation efficiency between kitplate DNA and chemically competent cells.

#### Notes

Put in Schrenk 128 incubator overnight.

#### Stop Time

10/23/2020

#### Next Time

The plates will be screened for transformed colonies through antibiotic resistance. If successful, if only one, still inoculate to get decent concentrations of DNA. Focus specifically on the RFP provided in the competent cell test kit.

## 11/1/2020 - 1:00 PM - Transformation

#### Start Time

1:00, 11/1/2020

#### Purpose

Transformation of a variety of different parts, including RFP, GFP, YFP, and competent cell test kit RFP. The difference here is the change to the competent test kit RFP. The team hypothesizes that there is a problem with kitplate transformations specifically. Debugging the transformation protocol is of the utmost importance, as getting high enough concentrations for restriction digest can not be completed without it.

#### Protocol

ig Sa Chemically Competent Cells Protocol (90 minute protocol)

* 4 x 50 uL chemically competent cells
* 1 ul of each plasmid DNA
* 950 uL of SOC broth for each Epi tube

#### Products

##### Transformation Epi Tubes

| Sample Label | Source Sample Label | Description |
| --- | --- | --- |
| 11/1/2020 CT1 | RFP BBa\_J04450 (kitplate) | 2018 KP4, Well 4B |
| 11/1/2020 CT2 | YFP BBa\_K1033910 | 2018 KP4, Well 6K |
| 11/1/2020 CT3 | RFP BBa\_J04450 (tube) | Competence Test Kit (iGEM) |

##### Petri Dishes

| Sample Label | Source Sample Label | Description |
| --- | --- | --- |
| 11/1/2020 CT1 1 50ul | 11/1/2020 CT1 | RFP BBa\_J04450 Control DNA on 9/3/2020 LB Agar (C) Plates |
| 11/1/2020 CT1 2 100ul | 11/1/2020 CT1 | RFP BBa\_J04450 Control DNA on 9/3/2020 LB Agar (C) Plates |
| 11/1/2020 CT1 3 100ul | 11/1/2020 CT1 | RFP BBa\_J04450 Control DNA on 8/25/2020 LB Agar Plates |
| 11/1/2020 CT2 1 50ul | 11/1/2020 CT3 | YFP BBa\_K1033910 on 9/3/2020 LB Agar (C) Plates |
| 11/1/2020 CT2 2 100ul | 11/1/2020 CT3 | YFP BBa\_K1033910 on 9/3/2020 LB Agar (C) Plates |
| 11/1/2020 CT2 3 100ul | 11/1/2020 CT3 | YFP BBa\_K1033910 on 8/25/2020 LB Agar Plates |
| 11/1/2020 CT3 1 50ul | 11/1/2020 CT4 | RFP BBa\_J04450 (tube) on 9/3/2020 LB Agar (C) Plates |
| 11/1/2020 CT3 2 100ul | 11/1/2020 CT4 | RFP BBa\_J04450 (tube) on 9/3/2020 LB Agar (C) Plates |
| 11/1/2020 CT3 3 100ul | 11/1/2020 CT4 | RFP BBa\_J04450 (tube) on 8/25/2020 LB Agar Plates |

#### Results

Control Tube RFP DNA led to successful growth while the kitplate DNA did not allow bacteria to grow on the plates. Therefore, the kitplate DNA transformations were not successful.

#### Notes

Put in Schrenk 128 incubator overnight.

#### Next Time

Focus on control RFP part versus kitplate RFP. These are the same parts, but one is the kitplate DNA while the other is sent inside the control tubes. Hypothesis that kitplate DNA protocol is being completed incorrectly leading to low to no transformations from any kitplate DNA.

#### Stop Time

4:15 pm 11/1/2020

## 11/1/2020 -  11:30 AM - Transformation

#### Start Time

11:30, 11/1/2020

#### Purpose

Transformation of a RFP and kitplate RFP. Debugging the transformation protocol is of the utmost importance, as getting high enough concentrations for restriction digest can not be completed without it.

#### Protocol

ig Sa Chemically Competent Cells Protocol (90 minute protocol)

* 4 x 50 uL chemically competent cells
* 1 ul of each plasmid DNA
* 950 uL of SOC broth for each Epi tube

#### Products

##### Transformation Epi Tubes

| Sample Label | Source Sample Label | Description |
| --- | --- | --- |
| 2/11/2020 CT1 | RFP BBa\_J04450 (kitplate) | 2019 KP1 23O RFP |
| 2/11/2020 CT2 | RFP BBa\_J04450 (tube) | Competence Test Kit (iGEM) |
| 2/11/2020 CT3 | Plain | Untransformed on LB plates |

##### Petri Dishes

| Sample Label | Source Sample Label | Description |
| --- | --- | --- |
| 2/11/2021 CT1 200ul C | 2/11/2020 CT1 | RFP BBa\_J04450 Control DNA on 9/3/2020 LB Agar (C) Plates |
| 2/11/2021 CT1 200ul LB | 2/11/2020 CT1 | RFP BBa\_J04450 Control DNA on 9/3/2020 LB Agar Plates |
| 2/11/2021 CT2 200ul C | 2/11/2020 CT2 | RFP BBa\_J04450 (tube) on 9/3/2020 LB Agar (C) Plates |
| 2/11/2021 CT2 200ul LB | 2/11/2020 CT2 | RFP BBa\_J04450 (tube) on 9/3/2020 LB Agar Plates |
| 2/11/2021 3 200ul C | 2/11/2020 CT3 | Untransformed on LB (C) plates |
| 2/11/2021 3 200 ul LB | 2/11/2020 CT3 | Untransformed on LB plates |

#### Results

Growth of control RFP DNA, however still no growth from kitplate plasmids. This means there is a definite problem with how the kitplate DNA is being used.

#### Notes

Put in Schrenk 128 incubator overnight.

#### Stop Time

3:00 pm 2/11/2020

#### Next Time

Broth culture of control RFP DNA to prepare dependent lab members for mini-prepping. Need more hands on the main project to make any meaningful progress.

## 4/10/2021 - Restriction Digest

#### Start Time

4/10/2021 4:26 pm

#### Purpose

Digest mutated chromoprotein composite part from the miniproject mutation PCR results. The point of this experiment is to teach dependent lab members the ligation protocol and the gel electrophoresis protocol once the digestions are completed.

#### Protocol

Standard iGEM double-digest procedure, with one Epi per the digestion. *Tube, labeled D1 and RBS + RFP + dT  37 degrees for an hour, then 80 degrees for 20 minutes*

* .2 mL PCR Epi tube
* MilliQ water, 19 µL
* 1x Tango Buffer, 2.5 µL
* DNA, 2 µL for 250 ng
* Enzyme 1: EcoR1, .5 µL
* Enzyme 2: Pst1, 1 µL

#### Products

| Sample Label | Source Sample Label | Description |
| --- | --- | --- |
| 4/10/2021 D1 | 4/1/21 2 PCR | Mutated RBS+RFP+dT from the results of the mutagenic PCR that the dependent lab members ran for the miniproject. |
| 4/10/2021 D2 | pSB1A3 2019 | Uncut Linearized plasmid backbone, provided in distribution |

#### Results

See results in the 4/11/2021 ligation and gel electrophoresis entry.

#### Notes

D1 followed protocol as detailed in the lab manual. D2 followed protocol as detailed on the iGEM linearized backbone protocol (found on the website). Because of this, the two digestions were not equivolume. Possible source of error when adding to the PCR machine.

#### Stop Time

4/10/2021

#### Next Time

Ligate digested parts together with miniproject dependent lab members. This step will technically be a loop, and standard assembly, as the chromoprotein part that we are working with is technically a composite part that includes the promoter. After ligation, run a gel electrophoresis on the mixture to check the results of the digestion and ligation [compare it against the DNA ladder included].

## 4/10/2021 - Transformation

#### Start Time

11:30, 11/1/2020

#### Purpose

Transformation of RFP composite part and pslr synthesized IDT parts. The purpose of this experiment is to test the effectiveness of transformation for parts not found in the kitplate, as tube RFP seemed to transform properly every single time it was tested. Therefore, external sources of DNA were acquired in order to better debug the procedure.

#### Protocol

ig Sa Chemically Competent Cells Protocol (90 minute protocol)

* 4 x 50 uL chemically competent cells
* 1 ul of each plasmid DNA
* 400 uL of SOC broth

#### Products

##### Transformation Epi Tubes

| Sample Label | Source Sample Label | Description |
| --- | --- | --- |
| 4/10/2021 CT1 | IDT BBa\_K117002 | IDT Synthesized pslrA part BBa\_K117002 |
| 4/10/2021 CT2 | IDT BBa\_K518012 | IDT Synthesized RFP BBa\_K518012IDT Synthesized RFP BBa\_K518012 |
| 4/10/2021 CT3 | RFP BBa\_J04450 (tube) | Competence Test Kit (iGEM) |

##### Petri Dishes

| Sample Label | **Source Sample Label** | **Description** |
| --- | --- | --- |
| 4/10/2021 CT1 100uL | IDT BBa\_K117002 | IDT Synthesized pslrA part BBa\_K117002 on AMP[Antibiotic] |
| 4/10/2021 CT2 100uL | IDT BBa\_K518012 | IDT Synthesized RFP BBa\_K518012 on KAN[Antibiotic] |
| 4/10/2021 CT3 100uL | RFP BBa\_J04450 (tube) | RFP BBa\_J04450 (tube) on [Chloramphenicol] plates |
| 4/10/2021 Control 1 | N/A | Control Plate with no transformation DNA [Kanamycin] |
| 4/10/2021 Control 2 | N/A | Control Plate with no transformation DNA, no antibiotic |

#### Results

Growth of IDT synthesized pslrA and RFP was witnessed on selection plates leading to a successful transformation.

#### Notes

Put in Schrenk 128 37° C incubator overnight.

#### Stop Time

4/10/2021

#### Next Time

Diagnose results of the transformation from the IDT synthesized parts and compare it against the tube RFP that always works.

## 4/11/2021 - Ligation

#### Start Time

4/11/2021 3:00 pm

#### Purpose

Ligate digested mutated chromoprotein with digested ampicillin backbone. Both parts were digested with EcoR1 and Pst1, meaning through standard assembly they should ligate together perfectly. This step is technically a loop, or one can view it as changing the antibiotic resistance of the mutated chromoprotein gene. Therefore, when plating the mutated chromoprotein make sure the antibiotic used is ampicillin as it will select against the unligated, undigested mutated chromoprotein part.

#### Protocol

https://www.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202Set up the following reaction in a microcentrifuge tube on ice:*Note)  Make sure to measure the concentration of DNA in the digestions before starting the procedure.*

1. T4 DNA Ligase Buffer (10X): 2 uL
2. Vector DNA: 50 ng
3. Insert DNA: 37.5 ng
4. Nuclease-free water: to 20 ul
5. T4 DNA Ligase: 1 uL

Mix by pipetting up and down

#### Products

| Sample Label | Source Sample Label | Description |
| --- | --- | --- |
| 4/11/2021 L1 | 4/1/21 2 PCR & pSB1A3 2019 | Standard ligation with two components: mutated chromoprotein composite part and plasmid backbone pSB1A3. Austin’s ligation reaction. |
| 4/11/2021 L2 | 4/1/21 2 PCR & pSB1A3 2019 | Standard ligation with two components: mutated chromoprotein composite part and plasmid backbone pSB1A3. Ben’s ligation reaction. |
| 4/11/2021 L3 | 4/1/21 2 PCR & pSB1A3 2019 | Standard ligation with two components: mutated chromoprotein composite part and plasmid backbone pSB1A3. Annika’s ligation reaction. |

#### Results

See results in the next step, running a gel to diagnose whether the PCR/digestion/Ligation was successful.

#### Stop Time

4/11/2021

#### Next Time

Run the three ligated products from 4/11/2021 (each of the three dependent lab members) on a gel to diagnose whether or not the previous trials were successful. Compared with the DNA ladder used. The chromoprotein is 1069 bp long and the backbone is 2155 bp long. In total, the band should be around 3224 bp long if properly ligated.

## 4/11/2021 - Gel Electrophoresis

#### Start Time

4/11/2021

#### Purpose

 Check to see if the PCR/ligation/digestions worked properly for the purpose of training dependent lab members. We will be using gel electrophoresis to test the length of the products through DNA ladder comparisons.

#### Protocol

Standard iGEM Gel Electrophoresis protocol, with made-from-scratch agarose gel.

#### Products

| Sample Label | Source Sample Label | Description |
| --- | --- | --- |
| Well 1 | 4/11/2021 L1 | 4/1/21 2 PCR & pSB1A3 2019. Ligation products from 4/11/2021 ligation [Austin]. |
| Well 2 | 4/11/2021 L2 | 4/1/21 2 PCR & pSB1A3 2019. Ligation products from 4/11/2021 ligation [Ben]. |
| Well 3 | 4/11/2021 L3 | 4/1/21 2 PCR & pSB1A3 2019. Ligation products from 4/11/2021 ligation [Annika]. |
| Well 4 | NEB DNA Ladder | [Insert kb] NEB DNA Ladder to measure the base length of the ligations through comparison. |

#### Stop Time

4/11/2021

#### Next Time

This will be the last step in the dependent lab member process unless the procedure works perfectly and the students wish to continue on with transformations to see what colors their mutations created.

## 4/18/2021 - Restriction Digest

#### Start Time

4/18/21 4:00 pm

#### Purpose

IDT Synthesized pslrA part BBa\_K117002 & IDT Synthesized RFP BBa\_K518012 for the purpose of designing a composite part with 3A assembly. These are our project parts, therefore the purpose of this digestion is to continue along with our project design.

#### Protocol

Standard iGEM double-digest procedure, with one Epi for the digestion. Cut reaction in half to preserve concentrations of mini-prepped parts.

#### Products

| Sample Label | Source Sample Label | Description |
| --- | --- | --- |
| 4/18/21 D1 BBa\_K117002 | 4/10/2021 CT1 | IDT pslrA part BBa\_K117002, E & S |
| 4/18/21 D2 BBa\_K518012 | 4/10/2021 CT2 | IDT RFP BBa\_K518012, X & P |
| 4/18/21 D3 psb1C3 | iGEM provided psb1C3 2019 | Linearized, uncut psb1C3, chloramphenicol standardized backbone, E & P |

#### Results

As detailed in the following gel purification step.

#### Notes

pslrA part was not digested due to missing Spe1 enzyme. First the chromoprotein IDT part will be digested and diagnosed with gel electrophoresis protocol. Same with the psb1C3 backbone. Instead of running protocol at 50 uL, a 25 uL alternative was instead picked. Miniprepped DNA concentrations were extremely low, leading the reaction concentrations to be skewed. Basically I used 250 ng of DNA in an otherwise normal 25 uL reaction (There should have been 500 ng in a 25 uL reaction).

#### Stop Time

4/18/21 5:00 pm

#### Next Time

Use gel electrophoresis to check the digestion of each of the products, and if working, use gel electrophoresis to purify each of the cut, linearized plasmids. Based upon the results of the digestion, determine whether or not you need to digest the promoter part. If the digestion is not working, only debug with one miniprep to save the amount of other miniprep. If digestion does not continue to work, try miniprepping the DNA again, except elute with 30 uL of buffer  instead of 100 to increase working concentration of each DNA miniprep.

## 4/19/2021 - Gel Electrophoresis

#### Start Time

4/19/2021

#### Purpose

Check to see if the digestions from 4/18/21 worked properly, specifically the IDT RFP and psb1C3 backbone parts. IDT RFP should be 828 bp long and the psb1C3 backbone should be 2070 bp long.

#### Protocol

Standard iGEM Gel Electrophoresis protocol, with made-from-scratch agarose gel.

#### Products

| Sample Label | Source Sample Label | Description |
| --- | --- | --- |
| Well 1 | NEB DNA Ladder | Standard DNA Ladder used to measure DNA length inside an agarose gel |
| Well 2 | 4/19/2021 D2 | IDT RFP BBa\_K518012, X & P with loading dye |
| Well 3 | 4/19/2021 D3 | Linearized, cut psb1C3, chloramphenicol standardized backbone, E & P with loading dye |

#### Results

Digests were not properly digested, with only single bands showing on the gel itself. However, because the DNA showed up on the gel, the concentration of using 250 ng is sufficient for diagnosing.

#### Stop Time

4/19/21

#### Next Time

The next step depends on the results of this procedure. If digestion works, buy Spe1 from NEB and digest the plsrA promoter used in our upcoming composite part. If the digestion doesn’t work, continue debugging the digestion until it works then proceed with the working protocol.

## 4/25/2021 - Restriction Digest

#### Start Time

4/25/2021

#### Purpose

Debug the digest protocol with the previous miniprepped parts. Dr. Westenberg recommended digesting each of the parts with a single enzyme to debug each enzyme individually. Therefore, standard digestion protocol will be used to digest the parts and then a gel will be run on the parts to test if proper digestion occurred.

#### Protocol

Standard iGEM single digest procedure, with one Epi for each digestion. Cut reaction in half to preserve concentrations of mini-prepped parts. Use 250 ng of DNA due to the relatively low concentrations of miniprepped parts.

Tube 1: IDT RFP BBa\_K518012, X & P [4/10/2021 CT2] 25 uL Reaction

1. 16.7 uL → 250 ng BBa\_K518012 [15 ng/uL]
2. 2.5 uL Cutsmart buffer
3. 0.5 uL Xba1 enzyme
4. 5.3 uL Milli-Q H2O

Tube 2: IDT RFP BBa\_K518012, X & P [4/10/2021 CT2] 25 uL Reaction

1. 16.7 uL → 250 ng BBa\_K518012 [15 ng/uL]
2. 2.5 uL Cutsmart buffer
3. 0.5 uL Pst1 enzyme
4. 5.3 uL Milli-Q H2O

Tube 3: Linearized psb1C3, E & P [psb1C3 2019] 25 uL Reaction

1. 10 uL → 250 ng psb1C3 [50 ng/uL]
2. 2.5 uL Cutsmart buffer
3. 0.5 uL EcoR1 enzyme
4. 17 uL Milli-Q H2O

Tube 4: Linearized psb1C3, E & P [psb1C3 2019] 25 uL Reaction

1. 10 uL → 250 ng psb1C3 [50 ng/uL]
2. 2.5 uL Cutsmart buffer
3. 0.5 uL Pst1 enzyme
4. 17 uL Milli-Q H2O

#### Products

| Sample Label | Source Sample Label | Description |
| --- | --- | --- |
| 4/24/21 D1 BBa\_K518012 | 4/10/2021 CT2 | IDT RFP BBa\_K518012, Xba1 |
| 4/24/21 D2 BBa\_K518012 | 4/10/2021 CT2 | IDT RFP BBa\_K518012, Pst1 |
| 4/24/21 D43 psb1C3 | iGEM provided psb1C3 2019 | Linearized, uncut psb1C3, chloramphenicol standardized backbone, EcoR1 |
| 4/24/21 D4 psb1C3 | iGEM provided psb1C3 2019 | Linearized, uncut psb1C3, chloramphenicol standardized backbone, Pst1 |

#### Results

As detailed in the following gel purification step.

#### Notes

First the chromoprotein IDT part will be digested and diagnosed with gel electrophoresis protocol. Same with the psb1C3 backbone. Instead of running protocol at 50 uL, a 25 uL alternative was instead picked. Miniprepped DNA concentrations were extremely low, leading the reaction concentrations to be skewed. Basically I used 250 ng of DNA in an otherwise normal 25 uL reaction (There should have been 500 ng in a 25 uL reaction). This should not be an issue as in the previous reaction 250 ng was sufficient for bands to show up on the gel.

#### Stop Time

4/25/2021

#### Next Time

Use gel electrophoresis to check the digestion of each of products with the single digestion, and if working, use gel electrophoresis to purify each of the cut, linearized plasmids. Based upon the results of the digestion, determine whether or not you need to digest the promoter part. If the digestion is not working, only debug with one miniprep to save the amount of other miniprep. If digestion does not continue to work, try miniprepping the DNA again, except elute with 30 uL of buffer  instead of 100 to increase working concentration of each DNA miniprep.

## 5/9/2021 - Gel Electrophoresis

#### Start Time

5/9/2021

#### Purpose

Check to see if the digestions from 4/24/21 worked properly, specifically the IDT RFP and psb1C3 backbone parts. IDT RFP should be 828 bp long and the psb1C3 backbone should be 2070 bp long. Look for results similar to single digestion results.

#### Protocol

Standard iGEM Gel Electrophoresis protocol, with made-from-scratch agarose gel.

#### Products

| Sample Label | Source Sample Label | Description |
| --- | --- | --- |
| Well 1 | NEB DNA Ladder | Standard DNA Ladder used to measure DNA length inside an agarose gel |
| Well 2 | 4/10/2021 CT2 | IDT RFP BBa\_K518012 Control |
| Well 3 | 4/24/21 D1 BBa\_K518012 | IDT RFP BBa\_K518012, Xba1 |
| Well 4 | 4/24/21 D2 BBa\_K518012 | IDT RFP BBa\_K518012, Pst1 |
| Well 5 | 4/24/21 D3 psb1C3 | Linearized, uncut psb1C3, chloramphenicol standardized backbone, EcoR1 |
| Well 6 | 4/24/21 D4 psb1C3 | Linearized, uncut psb1C3, chloramphenicol standardized backbone, Pst1 |

#### Results

Single bands on wells with “digested” parts. Therefore our DNA is of high enough concentration for visualization.

#### Stop Time

5/9/2021

#### Next Time

The next step depends on the results of this procedure. If digestion works, buy Spe1 from NEB and digest the plsrA promoter used in our upcoming composite part. If the digestion doesn’t work, continue debugging the digestion until it works then proceed with the working protocol for creating our composite RFP/plsrA project part via 3A assembly.

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# Team:Missouri Miners/Engineering

### ★ ALERT!

This page is used by the judges to evaluate your team for the [medal criterion](https://2021.igem.org/Judging/Medals) or  [award listed below](https://2021.igem.org/Judging/Awards).

Delete this box in order to be evaluated for this medal criterion and/or award. See more information at  [Instructions for Pages for awards](https://2021.igem.org/Judging/Pages_for_Awards).

# Engineering Success

### Silver Medal Criterion #1

Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.

Please see the [2021 Medals Page](https://2021.igem.org/Judging/Medals) for more information.

If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page) for your team to be eligible for this criteria.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Missouri Miners/Experiments

# 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.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Missouri Miners/Results

## Results

![](https://static.igem.org/mediawiki/2021/6/69/T--Missouri_Miners--results.png)

## Fluorometer Testing Trial 1

After developing the prototype fluorometer, a first round of response testing was completed to better understand the device’s response towards varying concentrations of RFP fluorescence. In the above figure, Lux was measured against varying inputs into the device. Trials began with a few baseline tests including “No Tube,” “Empty Tube,” and “Tube w/ H2O.” These trials were run to exhibit the behavior of the device with no sample. Next, a 10 fold serial dilution was performed on stock RFP to test how the device responded at varying concentrations of fluorescent particles and locate the limit of detection for the fluorometer. As detailed in the figure above, there was no significant response between the control tests and the sample runs, even at varying concentrations, when error is taken into account. This is likely due to possible light leakage in the device itself, bubbles in the sample, variations in the sample tube components and structure, and intensity issues with the LED used.