iGEM 2022 — Team umaryland

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## June

Lab notebook pdf, no notes needed

## July

Lab notebook pdf, no notes needed

## August

Lab notebook pdf, no notes needed

Three meetings with experts in the field of agriculture and environmental science were held this month. The first was with Dwight Dotterer from the University of Maryland Department of Agriculture. The second meeting was with Jose-Luis Izursa, a senior lecturer of Environmental Science and Technology at UMD. The final meeting was with Dr. David Ruppert, from the UMD College of Agriculture and Natural Resources. Fundraising for laboratory, collaboration, and miscellaneous expenses was launched this month. Emails and grant applications were sent out to ~20 different organizations by several team members. These organizations include several of the UMD departments, Chesapeake Bay conservation programs, and state/federal grants.

## September

The team applied for the iGEM impact grant in September. Fundraising was secured from the UMD Bioengineering department, and the impact grant was received later. Outreach meetings with Sarah Lane from the UMD center for Environmental Sciences and Ruth Cassily from the UMD Chesapeake Bay Agricultural Program were held. After our gene construct was cloned into bacteria, the results were sequenced. The results showed that the DNA from IDT had a deletion, and troubleshooting was required.

## October

Biochemical assay lab notebook pdf

Troubleshooting due to the deletion was completed and bacteria containing the final gene construct were propoduced. An educational meeting was held with the Howard Community Public High School Biotechnology Academy. Biochemical assays were run this month, validating the results of our cloning and that our gene construct is functional. The wiki was constructed by several team members.

## Wet Lab Notebook

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## Biochemical Assay Lab Notebook

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## Broad Overview of Design:

To reduce nitrates from the environment, we wanted the most efficient driver of nitrate dependent reactions we could find. This involved a few key features: first, the protein itself that would become our nitrate sink, then our promoter. The model essentially functions like this—if there is freestanding nitrate at an extreme excess in the environment, then within the bacteria, there must be a sink that collects the nitrate in some form that then becomes trapped and unable to return to the environment as free standing nitrate. This sink’s action will be driven forward by the promoter, while the promoter encourages the production of the protein next to it, the sink will be active. Eventually, the cell’s internal supply of nitrogen will run out, and to satisfy the demand created by the sink, the cell will intake the free nitrates in the environment.

## Issue and Fixes:

Our constructs were very large. When we first ordered them, we were made aware that though the constructs technically fit the parameters given by IDT, they were so bulky that they couldn’t make the full amount to give us. This didn’t technically cause any issues outright, but the constructs being big and unwieldy became a theme throughout the rest of the wetlab work.
cphA was so large that it was ordered in two pieces. cphB was marginally smaller. When we attempted to insert cphA1 and cphA2 into our plasmids and then transform into bacteria, we realized that only our cphB had been a success. However, since there was one small colony that seemed to be viable, we sampled from there and attempted to grow up our supply of cphA.
Eventually, once we managed to combine cphA with cphB and transform into bacteria, we got our DNA sequenced. We then realized that our DNA was not what we expected.
We witnessed a twenty base pair deletion in the middle of the sequence. We attempted to ensure that this was not an issue with our cloning; we redid the Gibson Assembly, retransformed, and witnessed the same issue over and over again. At this point, we assumed that something was wrong with the constructs we were given; we were, after all, informed from the beginning that for whatever reason, this construct seemed to stress IDT’s capabilities. To fix the issue, we used the Q5 Site Directed Mutagenesis Kit in order to repair our construct. We had to order corresponding primers to work with the kit. We performed the protocol once, transformed, and sequenced, then we realized that the first attempt wasn’t successful. However, once it was attempted a second time, the twenty base pair deletion had been fixed, determined through sequencing. We proceeded with Golden Gate, then encountered an overgrowth after transformation, which we suspected was due to old plates losing their antibiotic potency.

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## Research, Experiemnts, and Protocols

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#### Molecular Biology Protocols

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Luria Broth MediumAntibiotic SolutionsSOC MediumPermanent Strain StorageiGEM Transformation ProtocolNEB Gibson Assembly ProtocolPCR Reaction

#### Biochemistry Assay Protocols

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Nitrate Quantification Assay

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## Results:

This line excludes the first data point taken at 1M: it was obvious that the assay did not react appropriately at that concentration. Excluding that point, the correlation between absorbance and concentration was very strong and indeed very visible.

![](https://static.igem.wiki/teams/4483/wiki/image1.png)

(From left to right: 1M, 0.1M, 0.01M, 0.001M)

There was an odd outlier in our 0.01M LB solution, but barring that, which may have been due to error, all the samples were around 0.06 in absorbance.

Sample Wavelength Absorbance

LB 1 450 0.060613

LB 0.1 450 0.060613

LB 0.01 450 0.214448

LB 0.001 450 0.071711

This means that the absorbances noted for our samples can only be compared to each other.

Sample Wavelength Absorbance

cphA 1 450 1.986121297

cphA 2 450 1.130933166

cphA 3 450 0.557710588

cphB 1 450 1.323578715

cphB 2 450 0.383529276

Dud 1 450 1.34328568

Dud 2 450 0.918852449

Our most consistent data comes from the duds, neither of which was supposed to impact the nitrate concentration in the media. Unfortunately because of the LB standard curve not providing a clear translation from absorbance to concentration, it’s not possible to extrapolate from the absorbance to any particular strain’s impact on the media’s nitrate concentration. However, it is possible to note the consistency among the duds, the higher consistency between the cphA samples, and the relative chaos of the cphB samples.

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