iGEM 2018 — Team UFlorida

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# Team:UFlorida/Notebook

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[Public Engagement](https://2018.igem.org/Team%3AUFlorida/Public_Engagement)
[Entrepreneurship](https://2018.igem.org/Team%3AUFlorida/Entrepreneurship)

# Lab Notebook

### Week of 6/18/2018

* Isolated Plasmid pTRC99A
* Isolate Plasmid pTrc99A from *E. coli* using via Qiagen miniprep protocol.
* Streaked out Nissle 1917 on a plate

### Week of 7/1/2018

* The two PCR's performed failed due to bad plasmid stock
* Miniprepped fresh pKDsgRNA plasmid
* PCR reaction
* DpnI digestion
* PCR purification
* T4 Ligation\* with 2 hour incubation at Room Temperature
* Suspended plasmids from iGEM packaging
* Transformed 4 biobricks into Dh5 alpha cells
  1. BBa\_K1850010 (pSB1C3) fimH
  2. BBa\_K1850011 (pSB1C3) fimH
  3. BBa\_J04450 (pSB4K5) rfp
  4. BBa\_J04450 (pSB3T5) rfp* Transformation of the 4 pKDsgRNA plasmids into Dh5 alpha

### Week of 7/9/2018

* Isolated 136, 49, 3TS, 4KS via Qiagen miniprep protocol* Co-Electroporated biobricks in their respective plasmids (minipreppred from Dh5 alpha) into Nissle 1917 following electroporation protocol:
    + Combination 1: BBa\_K1850010 (pSB1C3) + BBa\_J04450 (pSB3T5)
    + Combination 2: BBa\_K1850011 (pSB1C3) + BBa\_J04450 (pSB3T5)* Transforming Competent Cells\* (Nissle WT)
    * Cultured transformed Nissle-pCas9-CR4 cells in LB broth for 4 hours
    * Transforming Competent Cells (Nissle\_pCas9-CR4)
    * Continuation of No-Scar
    * Make new Nissle and W3110 cultures: Repeat procedure from 7/11

### Week of 7/17/2018

* Transforming Competent Cells (Nissle with fimH 136KO or 49KO)
* Transforming Competent Cells (Nissle\_pCas9-CR4 transformed with pKDsgRNA-ldhA)
* Transforming Competent Cells (Nissle-fimH 49KO transformed with RFP-pSB3T5; Nissle-fimH 136KO transformed with RFP-pSB3T5)
* Transforming Competent Cells\* (Nissle-pkDldha-pcas9cr4 transformed with ldha oligo and ldha deletion oligo and control oligo)
* Assay for No-Scar and plating* Transforming Competent Cells\* (Nissle-fimH 49KO transformed with RFP-pSB3T5; Nissle-fimH 136KO transformed with RFP-pSB3T5)
  * Mutating ldhA gene
  * plasmid isolation of the 4K5 plasmid
  * Cultured Nissle in 9mL tubes for future fermentation (with and without phosphate buffer)
    + + Phosphate (0.9mL Buffer, 0.09mL Glucose (30%),8.01 mL LB)
    + -Phosphate (0.09mL Glucose (30%), 8.91 mL LB)* Incubator for 4 days
    * Made Glycerol stock culture of Nissle 1917, stored at -80 C
    * Culture Nissle and W3110 cultures
    * Run HPLC: -Nissle, -Nissle, +P, -Nissle, -P, -LB, -W3110
    + Look for presence of:Glucose, Succinate, Lactate, Formate, Acetate, Ethanol, Butyrate
    + Run HPLC and Fermentation on Nissle and W3110
    + HPLC Prep Protocol
    + Fermentation Prep Protocol

### Week of 7/23/18

* Run HPLC for Nissle fermentation: t=0,t=~20,t=48
* Re-ran HPLC after mixing samples.
* Detection of LDHA-Deletion Colonies by PCR
* Determine if culture genes have the deleted gene
* Fermentation Protocol
* Inoculated Nissle pCas9-cr4, Nissle 136 KO, fimH-pSB1C3, Nissle 49KO fimH-pSB1C3
* Inoculated Nissle pCas9-cr4 pKDsgRNA-ldhA
* PCR to detect the mutant strain
  + Troubleshooting: Why aren't the oligos for LdhA deleting the gene based on PCR results???
  + Hypothesis: Oligos are supposed to replace Okazoki fragments on the lagging strand. If Ori Right Replication Fork replicates faster than the left the LdhA gene will replicate on the opposite side!
  + Quick way to test hypothesis: Use a gene upstream of LdhA, AdhE, with appropriate oligos.

### Week of 7/30/2018

* Plate old iGEM parts in LB+chlor plates from DH5A.
  + K158 7000
  + K158 7004
  + K243 2000
  + K205 2016* Perform Transformation w/ Oligos for LdhA New (cDNA of Old oligo) and New+Old Oligos.
  * Plate iGEM written by RFP Nissile
  * Transform (pcas9cr4 nissle with pKD ldhA/adhe; wild type nissle with old parts (miniprepped, FimH 136 nissle with RFP)
  * Miniprep old parts (Shan's bacteria)
  * PCR to sequence our plasmids
  * Transforming pCas9cr4 with pKDsgRNA-AdhE and LdhA
  * Add the oligos (New & Both) to:
    + pCas9cr4 + pKDsgRNA-LdhA cells
    + Cas9 + pKD-AdhE, Cas9 + pKD-LdhA
    + ATC+ New
    + ATC+ Both
    + ATC- New
    + AtC- Both* Miniprep pKD46, pCP20, pKD4 from Shan's bacteria.
    * Miniprep old parts:K158 7000, K158 7004, K243 2000, K205 2016
    * Start of no-SCAR alternative pathway to delete genes.
    * Transformed by electroporation: K158 7005, K158 7004, PDK4, K205 2016, Nissle FimH KO 136 + rfp
    * miniprepped PKD46, PKD4, PCP20, K158 7005, K158 7004, K205 2016
    * streaked PCAS9 Cr4 + sgRNA\_LDHA and plated; PKD46, K205 2016, K158 7004, K158 7005, FimH 136KO + 4K5 Nissle on KAN
    * PCR amplified FRT-flanked resistant Kan gene with appropriate H1P1 and H2P2 frdA primers.
    * Screened colonies from the sgrna-ldha + pcas9cr4 (plated on chloramphenicol, induced with 188 nissle that had been "no-scar"ed with the new ldha oligo (lagging strand homology.)
    * screened 16 colonies that had been noscared with both the old and new ldha oligos (leading and lagging strand homologies)
    * Incubated pKDsgRNA-ldhA + pcas9cr4 to try no scar again

### Week of 8/6/2018

* Transformations, with 2 plasmids and 2 different strains: Nissle-pKD46, Nissle-pKDsgRNA, GM 272-pKD4, GM 272-pKDsgRNA
* Stitch fragments 1 and 2 of the butyrate fragment together via overlap extension PCR.
* Perform circular polymerase extension cloning into the amplified ptrc99A plasmid and the stitched together butyrate* fragment 1 and 2.
  * DNA Gel Extraction
  * Mini-prep
  * PCR on butryate fragment 2
  * Transformed WT Nissle with 46 pKD from miniprep
  * PCR of butyrate fragment 2 on gel
  * Transformations: Nissile with his pkD plasmid, Nissile with sgRNA

### Week of 8/13/2018

* PCR amplified Kanamycin FRT flanked gene on pDK with H1P1 and H2P2 primers WITH Q5 polymerase
* DNA purification
* Transformation
* Amplification of the butyrate downstream fragment (Nissle synthetic operon) with primers F1-But and R-CPEC, as well as the Q5 polymerase mastermix.

### Week of 8/20/2018

* Transformed rfp into k205 Nissle
* Transformed frdA oligos into Nissle pKDsgRNA-kan(frt) plated at 30 C on ATC to activate pCas9.
* Overlap Extension PCR assembly of butyrate fragment
* KOD Polymerase assembly of Butryate Fragment.
* Patching of bacterial colonies
* CPEC PCR Reaction
* Dh5 alpha was transformed with the butyrate biobrick cloned into ptrc99a plasmid
* PCR confirming the insertion of the butyrate biobrick into the dh5 alpha. Screening 5 colonies with KOD polymerase.
* Grew *E. coli Nissle* delta frdA with pkd46 plasmid
* Dpn1 DNA digest of ldha/Kan gene
* PCR Purified
* Electroporated ldha/Kan DNA and *E. coli Nissle* delta frdA cells with pkd46 plasmid.
* Transformation of pKDsgRNA and pKD46 with ldha oligos
* PCR Purification oligos(ldha,frda,adhe)
* PCR on Butyrate part 1 and Butyrate part 2
* Ran resulting fragments on a gel alongside restriction digested pieces
* Amplifying Butyrate Fragments
* CPEC Reaction (12.5uL) KOD polymerase + ptrc99a plasmid + butyrate part
* Incubate at 37 6 samples:(K205 2016, ldha- kan, Nissle frdA pkD46, K158 7005, pkD46 +ldha, K158 7004)
* HPLC protocol

### Week of 9/10/2018

* Screening butyrate CPEC transformed dh5-a colonies
* Screening KOD colonies with Onetaq
* Screened for butyrate insert with 3 different polymerases
* Transformed *E. coli Nissle* delta frda with the ldha - Kan fragment
* Crack and screen 100 Q5 colonies for a plasmid insertion into ptrc
* Transformed ptrc99A plasmid into dh5 alpha

### Week of 9/17/2018

* Screening
* Confirmed gene deletion of ldhA by pcr and gel

### Week of 9/24/2018

* Overlap PCR
* Butyrate fragment 1 amplification
* Nissle synthetic operon fragment amplification
* Cloning PCR for ter
* Gel purification
* VR screening of butyrate and fimH parts
* HPLC confirmed for ldhA deletion: no lactate produced: compared to model

### Week of 10/01/2018

* PCR screening for pcat ter
* PCR Screen But1 ptrc99a plate (Extend for 3kb)
* PCR Screen But2 ptrc99a colonies (Extend for 3kb)
* PCR Screen Full But Ptrc99A (extend for 5.5 KB)
* PCR Screen Full But - Pcat (extend for 5.5 KB)
* Miniprep
* Ter from Shan’s bacteria
* Cloning pBAD from part
* Fix “Ter from Shan’s bacteria” to:
  + Overlap Extension PCR of the Ter Gene from Dr. Shanmugam’s gift plasmid with the pBAD promoter. Attempted assembly into BBa\_K1587004
  + CPEC of the Ter gene with biobrick prefix and suffix into linearized psb1c3
  + Attempted insertion of the pBAD and pCAT promoters upstream of the Ter gene in psb1c3
  + PCR screen for Ter gene in pSB1C3, and for ter gene plus promoters in pSB1C3

### Week of 10/08/2018

* Continued PCR screening of colonies for parts cloned in the correct orienations
* HPLC analysis of transformed colonies for butyrate production.
* Experiment 1:
  + The Small Scale Fermentation Prep Protocol for Rotating Samples was run for two trials one being Wild Type E. coli Nissle (WT Nissle) and the other was E. coli Nissle with an ldhA gene deletion.
  + The Small Scale Fermentation Prep Protocol for Standing Samples was run for two trials one being Wild Type E. coli Nissle and the other was E. coli Nissle with an ldhA gene deletion.
  + Experiment 2:
    - The Small Scale Fermentation Prep Protocol for Standing Samples was run for ten total trials. Five were Wild Type E. coli Nissle and the other five were E. coli Nissle with an ldhA gene deletion.
    - HPLC protocol was carried out for all samples mentioned

### Week of 10/15/2018

* Experiment 1:
  + The Small Scale Fermentation Prep Protocol for Standing Samples was run for eight different samples ( E. Coli Nissle -ldhA + Butyrate Producing Part(BPP), BEM3 +BPP, Nissle -ldhA -frdA, Nissle 7004, Nissle -ldhA +7004, WT Nissle, BEM3 7004, and BEM3).
  + HPLC protocol was carried out for six samples ( E. Coli Nissle -ldhA + BPP, BEM3 +part, Nissle -ldhA -frdA, Nissle 7004, Nissle -ldhA +7004, WT Nissle) and a control (LB+arabinose+PO4).


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# Team:UFlorida/Experiments

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Engagement

[Human Practices](https://2018.igem.org/Team%3AUFlorida/Human_Practices)
[Public Engagement](https://2018.igem.org/Team%3AUFlorida/Public_Engagement)
[Entrepreneurship](https://2018.igem.org/Team%3AUFlorida/Entrepreneurship)

## Gene Deletion

We first attempted the no-SCAR (Scarless Cas9 Assisted Recombineering) method to remove ldhA (lactate dehydrogenase-A) from the E. coli Nissle genome. No-SCAR is different from the traditional CRISPR system used in eukaryotes because it uses CRISPR to counterselect against the wild type DNA, theoretically leaving only mutant colonies on the plate. Cells are transformed with two plasmids carrying Cas9 and the guide RNA. Cells are then transformed again with DNA oligonucleotides that confer a mutation to the target site of the guide-RNA. Colonies are incubated on antibiotic plates and mutant bacteria survive while wild-type are killed off by the CRISPR-Cas9 complex. E. coli naturally lack an efficient double-strand-break-repair system and CRISPR-Cas9 engineering can kill off wild-type cells after genetic targeting by the specific guide-RNA. This method takes advantage of the lack of repair and targets colonies that have failed to mutate (i.e. no gene deletion) and induces cell death.

### no-SCAR

![](https://static.igem.org/mediawiki/2018/d/de/T--UFlorida--noscar.png)

Diagram adapted from Reish 2015

However no-SCAR proved very difficult to manage in our specific *E. coli Nissle* chassis. Over one hundred bacterial colonies were screened for the deletion before we moved on to a different approach. This approach was not sustainable to rid one gene let alone the five carbon-draining genes in the short time we had left so we moved on.

![](https://static.igem.org/mediawiki/2018/3/30/T--UFlorida--deletionplates.jpg)

**Three groups: Control (C) Deletion (D) Mutation (M)**The Control group was transformed with an oligonucleotide homologous to a region of the genome outside of the guide RNA’s recognition (rpoB). The Deletion group was transformed with an oligo that caused a deletion of the entire *ldhA* gene. The Mutation group was transformed with an oligo that caused a stop mutation in the *ldhA* gene. Cas9 was induced via addition of anhydrous tetracycline on the right plate but not on the left, and 10-fold serial dilutions were made from the bottom of the plate to the top. No group had any order of magnitude of more killing than any other, implying that either recombination was not efficiently occurring or that crispr-Cas9 was not selectively killing wild-type bacteria as we had hoped.

We switched to the Datsenko-Wanner method. This technique utilizes antibiotic resistance replacement of the desired gene deletion. To accomplish this we used primers to amplify out a kanamycin resistance gene. This sequence was flanked upstream and downstream with regions of homology to the specific gene to remove. This amplified oligo was then used to recombineer a deletion and select for by kanamycin resistance. The resistance was necessary to remove so we used a FLP recombinase to 'flip' out the resistance gene. PCR and patching was used to confirm deletion. Within a week of switching, the *frd*  operon (fumarate reductase) was successfully deleted though dozens of colonies had to be screened in order to find the correct mutant. From then on this was our chosen deletion method.

### Datsenko-Wanner

![](https://static.igem.org/mediawiki/2018/e/e7/T--UFlorida--datsenko.png)

Diagram adapted from Datsenk and Wanner 2000

**1. Integrate Resistance Cassette**

![](https://static.igem.org/mediawiki/2018/e/ec/T--UFlorida--banddeletion.png)

#1. Ladder #2. Wild type (5kb) #3. frd operon + frt-kanamycin-frt cassette (1.5kb)

**2. Flip out Resistance Cassette**

![](https://static.igem.org/mediawiki/2018/7/7a/T--UFlorida--banddeletion2.png)

Frt-kanamycin cassette flipped out using the pcp20 plasmid encoded recombinase. The sample to the right of those two bands represents the previously integrated 1.5kb fragment.

The colonies with the confirmed removal of the Kan cassette were patched on kanamycin plates (pictured below). The plasmids we used, pCP20 and pKD46, had to be confirmed via patching that they're cured of the cell. This is done because unless the untransformed cell is sensitive for the antibiotic you're selecting it with, you wont be able to select for properly transformed cells. By plating the same colony on multiple antibiotic resistant plates, you can confirm that the cell is sensitive and resistant to respective antibiotics.

**3. Patching**

![](https://static.igem.org/mediawiki/2018/thumb/c/cb/T--UFlorida--patching.jpg/320px-T--UFlorida--patching.jpg)

However, we discovered through HPLC that fumarate was still being produced at higher concentrations than what was predicted by our metabolic model. We went back to the sequence and discovered that frdA is present in two copies in the *E. coli Nissle* genome. Although the gel showed the gene to be deleted, we had not known that a duplication existed.The second operon existed internally to the primers we used to screen and the homologous regions we made to delete with, so is was neither deleted nor detected. Two deletions would have been necessary to channel the carbon flow away from fumarate.

We decided not to go forward with that option as our model showed that other genes would have more carbon flux impact if deleted.

![](https://static.igem.org/mediawiki/2018/1/15/T--UFlorida--ldha_deletion.jpg)

Next to be deleted was *ldhA* which fortunately does not have a gene copy. This was validated by confirmation of fragment bands on a gel and lack of lactate production seen by HPLC measurements.

(Left) One colony had the 1.5kb kan-frt cassette integrated into its genome at the ldha location. Out of the 58 colonies screened for ldhA deletions, we found one that had it. Subsequent flipping out and loss of kanamycin resistance was successful.

### Butyrate Assembly

Team members optimized the five gene sequences for *E. coli Nissle 1917* for butyrate upregulation. The gene pathway was too long (5 kb) to be synthesized in one string so it was ordered as a gblock composed as two fragments. Synthesis was chosen over ordering the parts in a plasmid for cost efficiency. Butyrate fragment (BF) #1 contained atoB, hbd, and crt and BF #2 had ter and tesB. The ordered sequences were delivered with unintentional alterations that had extra copies of the Xbol digestion sites. This made it impossible to properly digest and ligate our gene fragments together, which is the typical way to assemble. A variety of other molecular techniques were attempted: (1) Overlap extension (2) Blunt Ligation of fragments #1 and #2.

The two fragments had regions that were similar to each other (overlapping) of about 40 bases each.Theoretically, if run through enough cycles of PCR the two fragments would become one due to elongation over the overlapping region (overlap extension). Even though a 5.3 kb band was visible after 12 cycles of overlap extension PCR, it was not possible to amplify this fragment to concentrations high enough to successfully assemble the part via Circular Polymerization Extension Cloning or restriction digestion into psb1C3 or other plasmids such as ptrC99A. This may have been due to poor primer binding, or due to the fact that our gBlocks were not ideal for PCR reactions.

We also attempted a blunt ligation of BF #1 and BF #2 into ptrc99A, to later be moved into psb1c3. These attempts also proved unsuccessful, as there were no bands in PCR reactions that screened for inserts.

### Overlap PCR

![](https://static.igem.org/mediawiki/2018/5/58/T--UFlorida--overlappcr.png)

### Circular Polymerase Extension Cloning

![](https://static.igem.org/mediawiki/2018/8/8c/T--UFlorida--cpec1.png)

After several months of work without any results, the Parts Registry and previous projects were searched for a solution. The Toulouse 2015 iGEM biobricks (BBa\_K1587004 and BBa\_K1587005) had four of the five enzymes contained in our gblock, but no ter. We attempted to improve the butyrate synthesis pathway BBa\_K1587004. A critical analysis of this pathway reveals that the *ccr* gene, which catalyzes the reduction of crotonyl-coA to butyryl-CoA, is NADPH-dependent. Without an enzyme that couples NADH to NADPH, this pathway will not be functional for butyrate production. Our initial rationale for gene deletions was to force *E. coli* to produce butyrate with our synthetic pathway in order to maintain redox balance, but this would be impossible without an enzyme that catalyzes the reduction using NADH instead of NADPH. For this reason, we attempted to add the trans-enoyl reductase (*ter*) to their pathway via restriction digestion to form a complete redox balanced pathway that could be driven further via gene deletions.

Thus, it was feasible to add our *ter* onto their assembly in order to complete the pathway. We successfully used PCR with primers containing the iGEM prefix and suffix to clone out *ter* from BF #2. This was followed by successful circular polymerase extension cloning into the psb1C3 backbone and transformation into *E. coli Nissle*, resulting in our first biobrick of *ter* without a promoter. We attempted to add the pCAT and pBAD promoters to this via 3A assembly from the distribution kit, but such attempts did not work and there was not enough time to troubleshoot before the parts deadline. We did confirm that our *ter*-psb1c3 part is RFC 10 compliant via restriction digestion, and have submitted it to the registry for future teams to use.

We did not stop trying. Below is our last attempt at assembling a functional butyrate pathway. This time.....we believe it worked!!! See Results/Demonstrate for more detail.

### Final Butyrate Pathway Assembly Method

![](https://static.igem.org/mediawiki/2018/2/27/T--UFlorida--final.png)

### FimH Addition

Red fluorescent protein (RFP) (BBa\_J04450) was inserted downstream of the engineered FimH via 3A assembly. This part was then moved back into pSB1C3 using Ecor-I and Pst-I digestion. PCR and restriction digestion were used to confirm that this part was RFC 10 compliant. Visual confirmation of the RFP gene expression is seen in the pictures below. Red-pink colonies appeared on a culture plate after several days of growth.

![](https://static.igem.org/mediawiki/2018/a/a7/T--UFlorida--rfp.png)

![](https://static.igem.org/mediawiki/2018/f/ff/T--UFlorida--rfppic.png)

Tube #1 - LB only

Tube #2 -wild-type  *E. coli Nissle*

Tube #3 -  *E. coli Nissle* transformed with FimH-RFP (in one plasmid)

Tube #4 -  *E. coli Nissle* transformed with RFP and FIMH (two separate plasmids)

Our FimH-RFP part BBa\_K267000 is clearly less "red" than FimH and RFP cotransformed onto separate plasmids. However our submitted part is useful in assays as seen below. This occurrence is likely due to genetic context dependency.

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

This test was performed on Caco-2 cells which are an immortal line of epithelial corectal cancer cells. Characterization assays showed that *E. coli Nissle* expressing our part were found closer to Caco-2 cells than colonies expressing RFP only, indicating that the Harvard Biodesign BBa\_K1580011 is intact and functional. Box (A) shows Caco-2 cells alone, and Box (B) and (C) show Caco-2 cells infected with engineered Nissle 1917. In Box (C) the 137 isoform failed to result in binding to Caco-2 cells. Box (B) shows the 49 isoform successfully bound to Caco-2 cells in suspension, concluding that the expression of RFP and fimH 49 did not interfere with each other, and robustly allowed for the visualization of engineered Nissle 1917 using our method. As an extra control, Box (D) shows wild type Nissle 1917 and Caco-2 cells. Physical binding was not seen in this group either.

![](https://static.igem.org/mediawiki/2018/e/e0/T--UFlorida--fimH.png)

Box (E) and Box (F) are the same cells but without prior addition of RFP by rhamnose. They show no binding because *fimH* is not being induced.This part was sequenced however the output was not added to the parts registry because we believe the data to be of poor quality due to improper primer binding. However the order of the composite part was confirmed by detection of FimH in many of the sequence reads near the beginning while RFP was not detected in the start region.

#### References

1. Reisch CR, Prather KLJ. Scarless Cas9 Assisted Recombineering (no-SCAR) in Escherichia coli, an Easy-to-Use System for Genome Editing. Curr Protoc Mol Biol. 2017 Jan 5;117:31.8.1-31.8.20. doi: 10.1002/cpmb.29.

2. Datsenko KA, Wanner BL.One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.
Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5

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# Team:UFlorida/Results

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# Results

✔ Deleted one gene copy of *frdA* from *E. coli Nissle 1917* genome

✔ Deleted *ldhA* from *E. coli Nissle 1917* genome

✔ Combined RFC and fimH BioBricks to create a detectable system of functional binding to colon cancer cells

✔ Submitted the *ter* gene in pSB1C3 to the registry

✔ Observed butyrate peak via HPLC after fermenting *LdhA/FrdA* deleted *E. coli Nissle 1917* strain transformed with a completed butyrate pathway.

Below is a graph of butyrate production by strains of Nissle with lactate dehydrogenase (*ldhA*) and one copy of fumarate reductase (*frdA*) deleted. We subtracted the amount of butyrate found in the media from these values. *Nissle – ldhA* + 7004 represents this same strain transformed with BBa\_K1587004, which provides a theoretical butyrate pathway that is not entirely NADH dependent and was created by the 2015 Toulouse iGEM team. *Nissle – ldhA* part represents this same deletion strain transformed with the butyrate part.

![](https://static.igem.org/mediawiki/2018/a/a5/T--Uflorida--adjustedbutyrate.png)

Based on these results, it appears that transforming the butyrate part and to some extent transforming BBa\_K1587004 into *E. coli Nissle 1917* with ldha deleted results in increased amounts of butyrate concentrations in the supernatant compared to wild type.

However, more samples need to be run in order to be statistically certain of this. Directed evolution can be employed to know for sure if the pathway has the potential to produce butyrate in greater titers. These will both be attempted by the jamboree. Deleting more genes, such as adhE, can increase the potential for butyrate production in this strain further and approach those that were observed in BEM3 plus the butyrate part.

Below represents the results of our engineered part transformed into BEM3, a strain given to us by Dr. Shanmugam to test the butyrate production by our part. BEM3 had several metabolite producing genes deleted, including *ldhA, adhE, frdA,* and others, but we did not delete these genes ourselves as this strain was given to us.

![](https://static.igem.org/mediawiki/2018/d/d9/T--Uflorida--adjustedbutyrate2.png)

Adjusted for the amount of butyrate naturally occurring in the media, it is apparent that our part is producing butyrate in this strain. More samples will be run to confirm the statistical significance of this finding, and directed evolution will be employed before the Jamboree to increase the titer of butyrate production. pH controlled fermentations will also be considered. BBa\_K1587004 did not produce any butyrate in this context, probably because it did not permit the survival of the strain under anaerobic growth (see OD’s).

In conclusion, our part appears to make butyrate and appears to be working better than BBa\_K1587004. BBa\_K1587004 appears to have a modest effect on butyrate production in *Nissle* with *ldhA* deleted, but more samples need to be run to increase confidence in the *Nissle* results.

# Future Directions

**Gene Deletions**

Our butyrate-producing system will have the most stoichiometric efficiency after all the genes we sought to delete have been deleted. After every deletion, we will apply the new information to our model to improve its efficiency, and with it, predict the effects of future gene deletions on our system.

**Directed Evolution**

In an attempt to maximize the butyrate production in our engineered system, directed evolution can be used to select for a more efficient pathway. Dr. Frances Arnold, who was awarded the Nobel Prize in chemistry this year, proved that you can co-evolve enzymes making up an entire biosynthetic pathway. Using a similar method, we could evolve our system to upregulate enzymes pertinent for butyrate biosynthesis.

**Testing**

With an engineered system that produces substantial amounts of butyrate, we can begin to start solving some of the problems which we sought to change.

*Probiotic* - With a bacterium that produces enough butyrate, we can patent and market our probiotic for preventative care of the gut microbiome.

*Therapeutic* - Our butyrate-producing probiotic will be co-transformed with our submitted part, RFP-FimH. This will allow us to explore the known apoptotic effects on colon cancer cells under a fluorescent microscope as previously shown in the project. From there, we can move on to in vivo experiments in mice to further the use of the bacteria.

*Microbes in Space* - Due to the advantages of microbes in space, showing our bacteria can be cultured in reduced gravity would prove useful in the case of using Nissle in space for its probiotic and industrial benefits.

**Part Submission**

There was no part submitted for the working butyrate production. This butyrate-pathway part will be registered as soon as digestion and PCR confirm that it is in the correct orientation and RFC10 compliant.

**Reflections**

Looking at how we could improve the project, we have concurred that individual enzyme upregulation would have been a better alternative. This would entail ordering the parts as the individual gene sequences (with promoter, RBS, terminator) and piecing them together one by one using 3A assembly. Assembly would be much simpler because there would be no lengthy sequences to amplify via PCR. This would significantly correspond to the butyrate metabolic model because every step would produce by-product concentration data to feed back into the model. Our model currently represents how each gene deletion affects the carbon flux, but it would benefit from a project design that added butyrate pathway enzymes one at a time. A more costly alternative would be to have the entire pathway synthesized as an IDT gene in a sequence verified plasmid.