iGEM 2019 — Team Florida

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# Team:Florida/Notebook

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[Design](https://2019.igem.org/Team%3AFlorida/Design)
[Experiments](https://2019.igem.org/Team%3AFlorida/Experiments)
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[Improve](https://2019.igem.org/Team%3AFlorida/Improve)
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[SAFETY](https://2019.igem.org/Team%3AFlorida/Safety)

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

[GLOSSARY](https://2019.igem.org/Team%3AFlorida/GLOSSARY)

AWARDS

[Model](https://2019.igem.org/Team%3AFlorida/Model)

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

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## Notebook

## Notebook


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# Team:Florida/Experiments

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[Parts Overview](https://2019.igem.org/Team%3AFlorida/Parts)

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

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

[GLOSSARY](https://2019.igem.org/Team%3AFlorida/GLOSSARY)

AWARDS

[Model](https://2019.igem.org/Team%3AFlorida/Model)

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

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# Experiments

### Workflow

The following procedure was repeated for all three components (SCRIBE, Cas9, pkdtrpoB565).
1. PCR amplification
2. Gel -> Clean-up (Miniprep) -> Dpn1: recognizes methylation sites from wild types
3. Cloning CPEC
4. Transformation + plating
5. Colony PCR to check
6. Work with plasmid with E. coli

### PCR amplification:

**Make solution of...**
1. Template: Cell Lysis (thermocycler) 98°C for 5 min
2. Nanopure water
3. Mastermix (Q5)
4. Primers
5. dnTPs
6. Magnesium
7. Buffer
8. Annealing temperature: 49°C

### Gel Electrophoresis

1. Mix PCR products with dye
2. Put ladder into gel
3. Put the mixture into the wells
4. Run gel at 100 volts, 30 minutes

### PCR Clean-up Miniprep

**Ethanol Purification**
1. Add 1uL Sodium Acetate
2. Sit in -20 °C overnight
3. Spin for 15 minutes to get DNA at bottom
4. Wash with 110uL 70% ethanol
5. Resuspend in EB
6. Place in -80°C

**Column Purification**
1. 1.5mL of each colony culture into tube
2. Centrifuge at max for 1 minute
3. Add another 1.5mL into each tube
4. Centrifuge at max speed for 1 minute
5. Resuspend pellet in 250uL buffer P1 and transfer to a microcentrifuge tube
6. Add 250uL of P2 mix thoroughly by inverting 4-6 times until solution becomes clear
7. Add 350uL of N3 and mix immediately by inverting.
8. Centrifuge for 10 min at 13,000 rpm
9. Apply supernatant from step 5 to Q1A prep spin column by pipetting. Centrifuge for 30-60 seconds & discard flow through the Q1A prep column and swittch off the vacuum source.
10. Add 500uL of PB Buffer
11. Wash spin column by adding 750uL PE buffer. Centrifuge for 30-60 seconds and discard flow. Transfer Q1A spin column to collection tube.
12. Centrifuge for 1 minute and remove residual wash buffer.
13. Place Q1A spin column in clean microcentrifuge tube.
14. To elute DNA, add 50uL EB buffer to column. Let it sit for 1 minute and centrifuge for 1 minute.

### Cloning (CPEC)

1. Anneal piece
2. Put CPEC (4uL ) into DH-alpha cells (Wait 30 min)
3. Water bath (45 seconds) 42°C
4. 2 minutes on ice
5. Add SOB
6. Recover for 1 hour (37°C incubator)
7. Plate on LBkan50 plate

### Transformation

1. Spin to get DNA to bottom of tubes
2. Add 1uL of plasmid A & B into other two tubes with cells (*E. coli* dHα)
3. Place 30 min on ice.
4. 42°C: heat shock in water bath for 45 sec
5. Add to ice for 2 min
6. Add 1 mL of SOB.
7. Incubate at 37°C for 1 hour
8. Spread culture using beads
9. Pipette 100uL onto plate
10. Spin tube for pellet
11. Re-suspend in media
12. Transfer liquid to plates
13. Incubate plates at 37°C

![](https://static.igem.org/mediawiki/2019/0/00/T--Florida--iGEM_plates_exp.jpeg)

### Electroporation

**Electroporation:** apply electrical pulse so that it pokes holes in the cell membrane. DNA can now enter the cell through those pores.

1. Water has other charges that will cause electrons to bind to them instead of cell membrane Make cells electrocompetent in order to complete electroporation
2. Add 1.5 mL BL21 to tube on ice
3. Spin at 4°C at 5 speed for 2 minutes to pellet the cells
4. Extract supernatant
5. Resuspend pellet in 500 uL cold nanopure, 500 uL glycerol mannitol
6. Spin for 2 minutes, vacuum liquid
7. Resuspend in 50 uL glycerol mannitol
8. Transfer to cuvette
9. Electroshock cuvettes
10. Suspend in SOB, transfer to tubes, incubate

### Colony PCR

-100 uL water into each well
-Put colonies into each well. Out tip into test tube with 5 mL SOB and 5 uL Kan
-Pick up 6 uL of water w colonies and put into the second strip of wells

### Serial Dilutions

**A. Culturing Tubes**
1. 5mL per tube of LB
2. 5uL of each antibiotic and IPTG (chlor40, spec100, kan50, and 0.1 mM IPTG)
3. Use stick to pick up colony and place in tubes
4. Put in 30°C incubator.

**B. Plating**
0 hr (no ATC)
10-3 to 10-8 dilution on non-selection plate
0 hours (3 replicates)
1st hour (3 replicates)
2nd hour (3 replicates)
3rd hour (3 replicates)

![](https://static.igem.org/mediawiki/2019/2/28/T--Florida--iGEM_colonies_exp_page.png)

### Comparing Rifampicin, LB, and Triple Antibiotics plates

1. Adding 180uL of H2O into each well of 96 well plate
2. Add 20uL of culture in first 3 wells
3. Add 20 uL from previous well into the one after to make 10^-1, 10^02 dilution, etc all the way up to 10^-6
4. Diluted 3 times in well plate
5. Place 10uL of each corresponding dilution onto all 6 plates using multichannel pipette.

**Incorporating rpoB in SCRIBE**
Firstly, we replaced the original target sequence on the SCRIBE plasmid pFF745 with the rpoB target sequence. Primers 2105, 2106, 2107, and 2108 were added to conduct a PCR amplification in which everything on the plasmid excluding the target sequence were amplified. Then, using Circular Polymerase Extension Cloning (CPEC), the rpoB sequence was attached to the pFF745 plasmid because the ends were complementary. The rpoB was obtained through purchase and contains some mutations on the sequence that will be transcribed later by the host *E. coli* cells. In this state, the plasmid is single stranded so Q5 master mix was utilized for PCR amplification in order to make the plasmid double stranded. We used Q5 because of its low error prone rate (1 in 1,000,000). Now, the plasmids are ready to be transformed into competent DH alpha E.coli cells. The cells were given a day to recover and then plated on agar plates with the corresponding antibiotic resistance: chloramphenicol. To distinguish between the cells with and without the new insert, colony PCR and gel electrophoresis was carried out. The forward primer binds to the target sequence while the reverse primer binds to the backbone. The forward primer only corresponds to rpoB therefore only cells with the new insert should show the corresponding band when performing gel electrophoresis. Lastly the cells with SCRIBE were induced with IPTG. rpoB encodes for the B subunit of the polymerase and contains mutations on the sequence that will be permanently incorporated into the *E. coli* cells through the SCRIBE system to give them rifampicin resistance. To test that SCRIBE, we plated the cultures induced with IPTG on rifampicin plates. If the cells grew, it was concluded that the SCRIBE system was working in the cells.

**Incorporating Cas9 and PkdtrpoB565**
For the next part, we are incorporating the CRISPR/Cas9 system onto separate plasmids to also be incorporated in the DH-alpha cells containing the SCRIBE system using electroporation. At first, we planned to incorporate the SCRIBE system, Cas9, and pkdtrpoB565 all on one plasmid but the cloning was the transformation portion was unsuccessful. At most, some cells contained only SCRIBE and Cas9 or SCRIBE and the guide RNA. To decipher which colonies had which combination we plated the cells containing kan/spec or kan/chlor. Then we transformed each cells with the missing component. We adjusted the procedure for additional replications to have each component cloned on separate plasmids.

The goal of this part of the project was to see whether the CRISPR/Cas9 system was as effective as the rifampicin antibiotic selection marker.

![](https://static.igem.org/mediawiki/2019/f/f6/T--Florida--exp4.png)

Source: Redman M, King A, Watson C, et alWhat is CRISPR/Cas9?Archives of Disease in Childhood - Education and Practice 2016;101:213-215.

We altered the promoter next to the reverse transcriptase component of the pFF745 to the promoter J23101. J23101 is a promoter synthetically designed to give high expression of the reverse-transcriptase sequence as well as the β-subunit sequence. The PkdtrpoB565 sequence was incorporated into the pFF745 plasmid and then transformed into the dH-alpha cells first. PkdtrpoB565 has spectinomycin resistance and encodes the guide sequence which binds complementary to the wild type sequence. Next, the Cas9 sequence was incorporated into the cells to probe for guide RNA bound to the wild type colonies. Cas9 then makes lethal double-stranded breaks to kill the wild types, leaving only the mutant colonies growing on the plates.

Note: Because both Cas9 and SCRIBE have chloramphenicol resistance, the SCRIBE plasmid was altered to have kanamycin resistance in order to differentiate between the three components.

Under the CRISPR/Cas9 system, ATC induces the tetracycline promoter which activates Cas9. The cells were split between plates with ATC and plates without ATC. Then for each set we made serial dilutions for the cells on plates with the three antibiotics (kan, spec, chlor), LB, and rifampicin to compare the efficiency rates based on plate count.

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# Team:Florida/Results

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TEAM

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PROJECT

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

PARTS

[Parts Overview](https://2019.igem.org/Team%3AFlorida/Parts)

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

HUMAN PRACTICES

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

[GLOSSARY](https://2019.igem.org/Team%3AFlorida/GLOSSARY)

AWARDS

[Model](https://2019.igem.org/Team%3AFlorida/Model)

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

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# Results

Our overall project can be broken down into three components. (1) Verifying the SCRIBE system (2) Compare the efficiency of the SCRIBE system against using Cas9 as a selection tool (3) Using the Van promoter to make the SCRIBE system more efficient.

## Verifying the SCRIBE system

For the first part of our project, we are testing to see if Fahim Farzadfard and Timothy K. Lu’s paper, “Genomically encoded analog memory with precise in vivo DNA writing in living cell populations” rings true in regards to whether the SCRIBE system works or not. In order to check for the SCRIBE system, we incorporated a target sequence that gave rise to specific antibiotic resistance. rpoB gives rise to rifampicin resistance and rpsL gives rise to streptomycin resistance. If the SCRIBE system is working correctly, then the target sequence was successfully incorporated into the chromosomal DNA of the *E.coli* cell. Therefore the host cell will express the corresponding antibiotic resistance.

# Rifampicin serial dilution plates

### Experiments 1-4

![](https://static.igem.org/mediawiki/2019/a/aa/T--Florida--rif_plate_101103.jpeg)

Dilution factor: 10-1 to 10-3

![](https://static.igem.org/mediawiki/2019/8/8b/T--Florida--rif_plate_104106.jpeg)

Dilution factor: 10-4 to 10-6

We checked to see if the rpoB target sequence was incorporated into the pFF745 plasmid and whether it gave the host cells rifampicin resistance. This was proved by the growth of colonies on the rifampicin plates.

## Experiment 1

The first experiment with E.coli represents the negative control therefore proving that pFF745 does not normally express rifampicin resistance.

## Experiments 2, 3, and 4

Experiments 2, 3 and 4 are all E. coli with the rpoB target sequence. Since they were able to grow on the rifampicin plate, we can conclude the SCRIBE system successfully worked.

The sequencing data shows that the rpoB target was successfully put into the plasmid. The sequencing reaction is on the bottom and the designed plasmid sequence is on the top. The gray bars at the top show that the whole sequence matches.

![](https://static.igem.org/mediawiki/2019/a/aa/T--Florida--rpob_sequence.png)

## Verifying Mutation is Caused by rpoB in SCRIBE only

**Goal: We wanted to verify the host cells’ resistant to rifampicin antibiotic came solely from the rpoB mutation and not from another source.**

rpoB is specific to rifampicin resistance while rspL is specific to streptomycin resistance. In order to rule out the possibility that other cells without the rpoB sequence can grow on rifampicin plates, we grew rpsL cells (with C and D orientation) on those plates. We patched the C and D cells on dilution plates, comparing cells that were and were not induced with IPTG (IPTG induces the SCRIBE system). As shown on the plate below, E. coli cells with rpsL as the target sequence were not able to grow on rifampicin plates. This supports our claim that only rpoB cells grew on the rifampicin plates.

![](https://static.igem.org/mediawiki/2019/a/a4/T--Florida--dpsl_cells.png)

DPSL cells with C and D orientation grown on Rifampicin plates

**Testing SCRIBE efficiency: Ideal time**

We wanted to test whether or not the amount of time we let the SCRIBE system run before inducing the system with ATC (to activate Cas 9 selection tool) made a difference in SCRIBE efficiency.

## Total Plate Counts:

![](https://static.igem.org/mediawiki/2019/6/6a/T--Florida--table_round_2.png)
![](https://static.igem.org/mediawiki/2019/d/d8/T--Florida--scribe_atc_time.jpeg)

In this experiment, we want to see if running the SCRIBE system for 24 hours vs. 48 hours made a difference in the system efficiency when induced with ATC. We induced the cells containing cas9 and the SCRIBE system with ATC. Then, we plated these cells immediately on the 0 hour column to test the growth of the cells without the cas9 system have time to run. After one hour of the cells being induced with ATC, we plated them and repeated this procedure after two and three hours of ATC inducement.

The graph confirms that over time, SCRIBE works more efficiently. This bolsters the results from our rpoB experiment as the data were similar. According to our data, the amount of time we let the SCRIBE system run affected how well the cas9 enzyme was able to kill off the wildtype cells. The longer we ran the SCRIBE/cas9 system, (48 hours of SCRIBE) the lower the colony count was at after 3 hours when compared to the shorter run (24 hours of SCRIBE). This indicates that over time, the longer SCRIBE had to work on the system, the more mutants were made and later degraded by the CRISPR/cas9 system.

## rpoB1 plates over time

![](https://static.igem.org/mediawiki/2019/e/ec/T--Florida--iGEM_rpob_48_hr.png)

rpoB1 (24 hours)

![](https://static.igem.org/mediawiki/2019/0/07/T--Florida--iGEM_rpob_24_hr.png)

rpoB1 (48 hours)

## rpoB2 plates over time

![](https://static.igem.org/mediawiki/2019/b/ba/T--Florida--iGEM_rpob2_24_hr.png)

rpoB2 (24 hours)

![](https://static.igem.org/mediawiki/2019/5/55/T--Florida--iGEM_rpob2_48_hr.png)

rpoB2 (48 hours)

## rpoB3 plates over time

![](https://static.igem.org/mediawiki/2019/a/a6/T--Florida--iGEM_rpob3_24_hr.png)

rpoB3 (24 hours)

![](https://static.igem.org/mediawiki/2019/e/e5/T--Florida--iGEM_rpob3_48_hr.png)

rpoB3 (48 hours)

## Efficiency of SCRIBE system vs. using Cas9 as a Selection Tool

Our team’s goal was to create a universal selection marker using the SCRIBE and CRISPR/Cas9 system. In this way, we can manipulate the SCRIBE system to incorporate any target sequence so that Cas9 can efficiently select for mutants that would otherwise be undetected by typical selection markers such as antibiotic resistance or color change.

## Counting Colonies

![](https://static.igem.org/mediawiki/2019/9/9c/T--Florida--iGEM_cas9-plate_colonies.png)

## SCRIBE without Cas9

![](https://static.igem.org/mediawiki/2019/c/c8/T--Florida--updated_scribe_nocas9.png)

## SCRIBE+Cas9 Plate Counts

![](https://static.igem.org/mediawiki/2019/3/3a/T--Florida--updated_scribe_cas9.png)

\*\*\*Highlighted regions indicate outlier (not included in “Plate type vs. Colony Count (9/17) graph)\*\*\*

![](https://static.igem.org/mediawiki/2019/8/8d/T--Florida--platetype_vs_colonycount.png )

We expected to see the plates that were induced with ATC (triple antibiotic and rifampicin plate) to have approximately the same amount of colony growth. This is because the triple antibiotic plate had only the cas9 as the selection tool while the induced rifampicin plate used both antibiotic resistance and the cas9 as selection tools. We believed that cas9 and antibiotic resistance would have similar efficiencies in killing wild type cells (non-mutants). According to our graph, we see that the cas9 and triple antibiotic plate had around the same number of colonies. Thus, we can support our hypothesis that cas9 works as a selection tool.

When we use Cas9 as a selection tool (as demonstrated by the triple antibiotic plate), we see that there are more colonies than on the induced rifampicin plate. Thus, it is possible that some wildtype colonies left on the plate when we use cas9 as opposed to using it in conjunction with rifampicin resistance (given by the SCRIBE system). This leads us to conclude that Cas9 can be used as a selection tool, but it is not as efficient when coupled with the SCRIBE system.

The uninduced LB and rifampicin plate did not contain ATC. Therefore, cas9 was not working on these plates. The LB plate showed significantly more colony growth since there was no selection present on the plate. Both wildtype and mutant cells were grown on LB plate. The rifampicin plate has fewer colonies growing because the antibiotic acts as the selection tool.

### Van Promoter Increases Production of Beta Protein

When SCRIBE is turned on, it produces ssDNA. Beta proteins bind to the ends of the DNA, aiding in site specific homologous recombination of the sequence into the Okazaki fragments during lagging strand synthesis. They also protect ssDNA from degradation in the cell since the host cell would read the ssDNA as foreign DNA. Sometimes during transcription, the RNA polymerase will not reach the Beta subunit, leading to the early termination of transcription. This makes the system less than optimal because the beta subunit will not always be transcribed during replication. To prevent this, we incorporated the Van promoter to be in front of the B-subunit sequence, improving the overall efficiency of the SCRIBE system. Now, each component of the plasmid has its own promoter.

## Experiments:

1. E. coli
a. Plated E. coli on LB and Rifampisin plates to act as negative control
2. Only SCRIBE system working (with Lac promoter)
a. Wanted to test for and see the current efficiency of the SCRIBE system
3. Lac promoter and van promoter
a. Testing to see if the van promoter make the SCRIBE system more efficient
4. J23 promoter and van promoter
a. Testing to see if replacing the Lac promoter with J23 promoter increased efficiency
b.Working alongside the van promoter

### LB plates (Experiments 1-4)

![](https://static.igem.org/mediawiki/2019/3/33/T--Florida--lbplate1.png)

LB Plates Dilution factor: 10-1 to 10-3

![](https://static.igem.org/mediawiki/2019/e/e7/T--Florida--lbplate2.png)

LB Plates Dilution factor: 10-1 to 10-3

## Rifampicin plates (Experiments 1-4)

![](https://static.igem.org/mediawiki/2019/a/aa/T--Florida--rif_plate_101103.jpeg)

LB Plates Dilution factor: 10-1 to 10-3

![](https://static.igem.org/mediawiki/2019/8/8b/T--Florida--rif_plate_104106.jpeg)

LB Plates Dilution factor: 10-1 to 10-3

From this we can see that the E.coli was able to grow on the LB plates but not able to grow on the Rifampicin plates (Experiment 1). The SCRIBE system was more efficient when the van promoter was introduced into the system as evident from the difference in the colony counts for experiments 2 vs 3&4 (2 was only the SCRIBE system working while 3&4 had the van promoter working as well). We wanted to see if the J23 promoter worked better in place of the Lac promoter the J23 is a high efficiency promoter. On the rifampicin plate, we see that there was more colony growth with the J23 promoter than with Lac (Experiment 4 and Experiment 3 respectively). Both systems also had the van promoter in it which supports the claim that the van promoter increases SCRIBE efficiency.

# Project Achievements

### Successful Results

* Incorporating RpoB target sequence into SCRIBE
* Incorporating SCRIBE into DH5-alpha cells resistant to rifampicin
* Incorporating cas9 into pFF745
* Modifying pFF745 from chloramphenicol to kanamycin resistance
* Incorporating pkdtrpob565 into pFF745
* Incorporating SCRIBE, cas9, and pkdtrpob565 into DH5-alpha cells
* Using cas9 as a selection marker against wild type cells
* Incorporating J23101 promoter in front of reverse transcriptase
* Van promoter

+ Van promoter was initially not successfully incorporated into the pFF745 plasmid
+ To confirm this we sequenced the cells and found that the van promoter was not successfully incorporated into the plasmid.
+ Therefore, we performed another transformation which successfully incorporated the Van promoter into the plasmid (PCR amplify the PvanCC promoter and the PFF backbone, and CPEC the two pieces together)

### Unsuccessful Results

Column chromatography did not work when purifying DNA. Instead we used ethanol purification. This was an alternative DNA purification method to purify Van promoter because the column chromatography did not work.