iGEM 2021 — Team Groningen

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# Team:Groningen/Notebook

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Groningen 2021

* Project
  + [Description](https://2021.igem.org/Team%3AGroningen/Description)
  + [Model](https://2021.igem.org/Team%3AGroningen/Model)
  + [Contribution](https://2021.igem.org/Team%3AGroningen/Contribution)
  + [Implementation](https://2021.igem.org/Team%3AGroningen/Implementation)
  + [Awards](https://2021.igem.org/Team%3AGroningen/Medals_and_Awards)
* Wet lab
  + [Engineering](https://2021.igem.org/Team%3AGroningen/Engineering)
  + [Notebook](https://2021.igem.org/Team%3AGroningen/Notebook)
  + [Experiments](https://2021.igem.org/Team%3AGroningen/Experiments)
  + [Results](https://2021.igem.org/Team%3AGroningen/Results)
  + [Parts](https://2021.igem.org/Team%3AGroningen/Parts)
  + [Safety](https://2021.igem.org/Team%3AGroningen/Safety)
* People
  + [Team](https://2021.igem.org/Team%3AGroningen/Team)
  + [Attributions](https://2021.igem.org/Team%3AGroningen/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AGroningen/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AGroningen/Partnership)
  + [Sponsors](https://2021.igem.org/Team%3AGroningen/Sponsors)
* Outreach
  + [Human
    Practices](https://2021.igem.org/Team%3AGroningen/Human_Practices)
  + [Integrated Human
    Practices](https://2021.igem.org/Team%3AGroningen/Gold_Human_Practices)
  + [Education](https://2021.igem.org/Team%3AGroningen/Communication)

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# Notebook

## Introduction

On this page we have documented our weekly wet lab progress during the iGEM season. The purpose of the Notebook is to outline which experiments were done when and who conducted them, and also to present some of the preliminary results.
To see the final results in detail, please head over to the **[Results page](https://2021.igem.org/Team%3AGroningen/Results)**. To learn more about our engineering approach, head over to the
**[Engineering page](https://2021.igem.org/Team%3AGroningen/Engineering)**.

## Calendar

Calendar

Lab Weekly Calendar

Weekly Summary

Previous Week

Next Week

#### iGEM Groningen

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#### Project

* [Description](https://2021.igem.org/Team%3AGroningen/Description)
* [Model](https://2021.igem.org/Team%3AGroningen/Model)
* [Contribution](https://2021.igem.org/Team%3AGroningen/Contribution)
* [Implementation](https://2021.igem.org/Team%3AGroningen/Implementation)
* [Medals](https://2021.igem.org/Team%3AGroningen/Medals_and_Awards)

#### Wet lab

* [Engineering](https://2021.igem.org/Team%3AGroningen/Engineering)
* [Notebook](https://2021.igem.org/Team%3AGroningen/Notebook)
* [Experiments](https://2021.igem.org/Team%3AGroningen/Experiments)
* [Results](https://2021.igem.org/Team%3AGroningen/Results)
* [Parts](https://2021.igem.org/Team%3AGroningen/Parts)
* [Safety](https://2021.igem.org/Team%3AGroningen/Safety)

#### People

* [Team](https://2021.igem.org/Team%3AGroningen/Team)
* [Attributions](https://2021.igem.org/Team%3AGroningen/Attributions)
* [Collaborations](https://2021.igem.org/Team%3AGroningen/Collaborations)
* [Partnership](https://2021.igem.org/Team%3AGroningen/Partnership)
* [Sponsors](https://2021.igem.org/Team%3AGroningen/Sponsors)

#### Outreach

* [Human practices](https://2021.igem.org/Team%3AGroningen/Human_Practices)
* [Integrated Human practices](https://2021.igem.org/Team%3AGroningen/Gold_Human_Practices)
* [Education](https://2021.igem.org/Team%3AGroningen/Communication)

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#### We thank our sponsors:

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# Team:Groningen/Engineering

[![](https://static.igem.org/mediawiki/2021/1/17/T--Groningen--LogoByeMonia.png)](https://2021.igem.org/Team%3AGroningen)

Groningen 2021

* Project
  + [Description](https://2021.igem.org/Team%3AGroningen/Description)
  + [Model](https://2021.igem.org/Team%3AGroningen/Model)
  + [Contribution](https://2021.igem.org/Team%3AGroningen/Contribution)
  + [Implementation](https://2021.igem.org/Team%3AGroningen/Implementation)
  + [Awards](https://2021.igem.org/Team%3AGroningen/Medals_and_Awards)
* Wet lab
  + [Engineering](https://2021.igem.org/Team%3AGroningen/Engineering)
  + [Notebook](https://2021.igem.org/Team%3AGroningen/Notebook)
  + [Experiments](https://2021.igem.org/Team%3AGroningen/Experiments)
  + [Results](https://2021.igem.org/Team%3AGroningen/Results)
  + [Parts](https://2021.igem.org/Team%3AGroningen/Parts)
  + [Safety](https://2021.igem.org/Team%3AGroningen/Safety)
* People
  + [Team](https://2021.igem.org/Team%3AGroningen/Team)
  + [Attributions](https://2021.igem.org/Team%3AGroningen/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AGroningen/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AGroningen/Partnership)
  + [Sponsors](https://2021.igem.org/Team%3AGroningen/Sponsors)
* Outreach
  + [Human
    Practices](https://2021.igem.org/Team%3AGroningen/Human_Practices)
  + [Integrated Human
    Practices](https://2021.igem.org/Team%3AGroningen/Gold_Human_Practices)
  + [Education](https://2021.igem.org/Team%3AGroningen/Communication)

![](https://static.igem.org/mediawiki/2021/b/b8/T--Groningen--EngineeringBanner.png)

# Engineering

* [Improving alpha-amylase production](#impr)
* [Design overview](#design)
* [Cloning strategy design](#clone)
* [Cloning pipeline](#pipe)
* [Production of alpha-amylase](#prod)
* [Combinatorial library - Subset](#poss)
* [References](#ref)

On this page the genetic engineering strategy to produce alpha amylase can be found. An overview of how alpha amylase has been integrated in our constructs, expressed in *Saccharomyces spp.* and measured can be found in **Figure 1.**

![workflow](https://static.igem.org/mediawiki/2021/c/cf/T--Groningen--ExperimentsWorkflowNEW.png)

***Figure 1.**Steps that were taken to express and measure alpha amylase in *Saccharomyces spp.* First, the optimized coding sequence was inserted into an entry vector (pYTK001), and amplified in *E. coli*. After that, the final constructs were assembled and amplified. Finally, alpha amylase was expressed in *Saccharomyces spp.* Quantitative and qualitative measurements on alpha amylase activity were taken, more on that in **Figure 9.***

## Improving alpha-amylase production

The main engineering challenge our team is working on this year is producing the **most functional alpha-amylase enzyme**. We characterised the activity of four different alpha-amylase genes, originating from 4 host organisms,
*Bacillus amyloliquefaciens***[1]**, *Bacillus licheniformis***[1]**, *Bacillus subtilis***[1]** and *Aspergillus oryzae***[1]**. We chose these enzymes as they are well reviewed and annotated, furthermore, they have proved to work in similar conditions to the ones found in the cow’s rumen. These genes were
expressed in the chassis *Saccharomyces cerevisiae* and *Saccharomyces paradoxus*, as it can feed in ammonia as a only nitrogen source, an essential part of our project (see more on **[Description page](https://2021.igem.org/Team%3AGroningen/Description)**).

Modularity is a very important feature of our engineering process, as this year we are dealing with a lot of different variables in each genetic element and we wanted them to be able to be easily interchangeable. In order to optimize
the Design-Build-Test-Learn cycle (DBTL cycle) we built a selected library of constructs out of the full spectrum of possible combinations and we used AI technology to learn from the set of samples.

## Design overview

The genetic elements present in our construct are a **promoter** (Part 2), **two different versions of each alpha-amylase gene** (Part 3); one consisting of the **native sequence** that also codes for the secretion peptide (SP) and one in which the SP has been **engineered and substituted** by characterized yeast SPs, finally the **terminator** (Part 4) is added. The basic configuration of the constructs can be found in **Figure 4.**

The Parts containing the 4 different alpha-amylase genes were designed according to the Golden Gate MoClo Assembly technique. The 4 different alpha-amylase genes were built to be **Part 3** and **Part 3b** according to the MoClo assembly strategy as can be seen in **Figure 3**, this part refers to the coding sequence. The genetic design can be seen in **Figure 5** and **6**. Additionally, various promoters and terminator from MoClo Toolkit (Kit #1000000044) were used. By using these different promoters we tried to find the best way to express our gene of interest. An overview of the different promoter and alpha-amylase parts used can be found in **Table 1**. The backbone vector pRS426\_\_ConLS'-GFPdropout-ConRE'-URA3-2micron-Kan was used in all the constructs (**Figure 2**). The vector contains Parts 1 and 5 - 9 of the kit, to see the full composition of the vector and the components of it check **Figure 3**. Furthermore, the vector contains selection markers for both *E.coli* and *Saccharomyces spp.* (kanamycin and URA marker respectively), and a GFP drop out which facilitates the screening of successful assemblies.

![Backbone used as a vector for all the constructs tested.](https://static.igem.org/mediawiki/2021/6/60/T--Groningen--Picture1.png)

***Figure 2.** Backbone used as a vector for all the constructs tested. GFP dropout was used to screen for colonies that took in the correct plasmid.*

![Different Parts plasmid included in the MoClo kit (Lee M et al, 2015) and engineering strategy.](https://static.igem.org/mediawiki/2021/0/06/T--Groningen--PartPlasmids.png)

***Figure 3.** Different Parts plasmid included in the MoClo kit**[2]** and engineering strategy. We used a backbone plasmid containing Part 1, Part 5, Part 6, Part 7, Part 8, and Part 9. We then cloned Part 2, Part 3 (or 3a and 3b) and Part 4.*

![Diagram displaying the different elements of the genetic construct.](https://static.igem.org/mediawiki/2021/a/a5/T--Groningen--GeneticConstruct.png)

***Figure 4.** Diagram displaying the different elements of the genetic construct. As we are using the Golden Gate MoClo Assembly technique. The number refers to the corresponding part of the cloning technique. **A)** Construct with the native sequence of the alpha amylase, **B)** Construct where the native SP has been substituted by a yeast secretion peptide from**[3]**.*

## Cloning strategy design

### Part 3

The 4 different native alpha-amylases were designed in a similar way, we obtained the sequences from the Uniprot database and optimized them for our cloning goals. We codon optimized the original sequence for our chassis and
we added the corresponding overhangs to prepare it for the **Golden Gate Assembly technique**. In order to do that, we added some sequences up and downstream the genes. These sequences correspond to the recognition
sites of the **restriction enzymes type IIS BsmBI and BsaI** (**Figure 5**).Using two different restriction enzymes allows for a two steps cloning strategy. Firstly, integration of the new design part (alpha amylase genes) into an *E.coli* vector, pYT001, by a digestion and ligation protocol with **BsmBI**. This way the new part is already assembled in a vector and can be easily multiplied. Every Part of the kit is assembled in this type of vector, which has a chloramphenicol marker so that the parts can be amplified in *E. coli*. Next, the **BsaI** restriction enzyme is used to assemble the Part plasmids together , in what is referred to as cassette plasmid. The cassette plasmid is first amplified in *E.coli*, before downstream cloning in the desired chassis; *Saccharomyces spp.* More information on this below (**[Cloning pipeline](https://2021.igem.org/Team%3AGroningen/Engineering#pipe)** )

Once the sequence was designed the genes were ordered and synthesized ***de novo***.

![Overhangs of alpha amylase gene, Part 3 of the final vector.](https://static.igem.org/mediawiki/2021/6/6f/T--Groningen--Overhangs.png)

***Figure 5.** Overhangs of alpha amylase gene, Part 3 of the final vector. The sequences were added in the four alpha amylases genes.*

### Part 3b

Part 3b was designed based on Part 3, using primers to amplify the desired sequence from the *de novo* synthesized alpha amylases. The aim of this part is to code for the alpha-amylase protein, **excluding the native secretion signal**. The idea behind this exclusion is adding more variability to the library of samples created, as the secretion signal has been proven to be a **bottleneck** in the production of a secreted enzyme**[4]**. Secretion
peptide sequences are well annotated, therefore, identifying them wasn’t an arduous task. We designed primers that will only amplify the desired part of the alpha-amylase sequence and add the sites of the enzymes of interest
(BsmBI and BsaI). The primers designed can be seen in **Figure 6**.

![Design of the primers used to amplify Part 3 and create Part 3b.](https://static.igem.org/mediawiki/2021/0/0b/T--Groningen--Primers.png)

***Figure 6.** Design of the primers used to amplify Part 3 and create Part 3b.*

The creation of Part 3b involved one PCR reaction amplifying only the secreted enzyme sequence, therefore, before using the part for downstream cloning steps, PCR results needed to be **verified**. To do so, an 1% agarose gel electrophoresis analysis was performed (**Figure 7**). In addition, to make sure no mutations occurred during the cloning process the entry vector was sequenced. (see the **[Results page](https://2021.igem.org/Team%3AGroningen/Results)** ).

![Agarose gel from PCR to amplify Part 3b from Part 3.](https://static.igem.org/mediawiki/2021/8/89/T--Groningen--AgaroseGel.png)

***Figure 7.** Agarose gel from PCR to amplify Part 3b from Part 3. The sizes are the ones expected.*

## Cloning pipeline

For understandability purposes an overview of the cloning pipeline is summarised below.

Part 3 and Part 3b were inserted into the **vector pYT001**, the “entry vector”, that contains a resistant marker for **chloramphenicol**, so the vector can be amplified in *E. coli*.
This insertion was done in a Golden Gate MoClo Assembly with **BsmBI**. After that, we performed a Golden Gate MoClo Assembly with **BsaI** where all the parts were included, part plasmid for Part 2 (promoters), Part 4 (terminators) and
Part 3a (secretion signals) was taken from the kit. *E. coli* was also used to amplify the product before transforming our chassis, *Saccharomyces spp.* A visual explanation of this can be seen in **Figure 8.**

![Cloning pipeline to create the library of constructs.](https://static.igem.org/mediawiki/2021/4/4e/T--Groningen--CloningPipeline.png)

***Figure 8.** Cloning pipeline to create the library of constructs. In the first Assembly BsmBI is used, while in the second digestion, BsaI was added.*

## Production of alpha-amylase

To prove that we are producing a heterologous **functional enzyme** in *Saccharomyces spp.*we decided to use different assays, one of them being a**starch assay**. Starch
is broken down into smaller saccharides by the enzyme alpha-amylase. By adding starch to the media and checking whether it is being degraded we can measure if alpha-amylase is being produced and, what is even more important,
whether it is functional. Production and functionality were also tested through the **alpha-amylase kit assay**. This assay provided quantitative data from high throughput experiments. Illustrations on how alpha amylase is measured can be found in **Figure 9**. To find out detailed protocols on how to perform the experiment go to the [Experiments page](https://2021.igem.org/Team%3AGroningen/Experiments)
![kitassay](https://static.igem.org/mediawiki/2021/3/35/T--Groningen--Engineering_kitassay1.png)
![halotest](https://static.igem.org/mediawiki/2021/a/aa/T--Groningen--Engineering_halotest1.png)

***Figure 9.** Alpha amylase production was evaluated through two assays. a) **Kit assay**, it is a quantitative assay that uses colorimetric output to measure the alpha amylase activity, more information on how the kit works can be found in the Experiments and Results page. b) **Halo test**, qualitative essay through which the cleavage of starch by alpha amylase can be assessed.*

On this page, evidence about the performance of basic parts can be found, for extended results go to the [Results page](https://2021.igem.org/Team%3AGroningen/Results).

### Part 3

We created a library from all the possible combinations and based on preliminary results some of the samples were tested in the halo test. Sample SP027, which was composed by the promoter pRNR1, **Part 3 from** ***B. Subtilis*** and tTDH1 was expressed in the yeast ySB76 (*S. cerevisiae*). We used different methods to check for the intracellular alpha-amylase and compare it, results can be seen in **Figure 10**, detailed protocol can be found
on the [Experiments page](https://2021.igem.org/Team%3AGroningen/Experiments). Overall, the halo is not visible when you use the cells, and it increases when plating the supernatant of liquid cultures (where
alpha-amylase is supposed to be found), even more when cells are lysed previously. The latest result shows that **alpha-amylase** from *B. subtilis* is present in both the supernatant and inside the cells.

![Triplicates of halo test in sample SP027, alpha-amylase from B.subtilis.](https://static.igem.org/mediawiki/2021/b/b8/T--Groningen--Engineering_Figure8.png)

***Figure 10.** Triplicates of halo test in sample SP027, alpha-amylase from B.subtilis. Different products of the cells were used to compare the activity, **a)** Yeast cells, **b)** cells’ supernatant from liquid cultures and **c)** lysate from cells by bead beating them during 10s.*

Through the colorimetric assay which is based on the same principle we could prove that the other Part 3 from *A. oryzae*, *B. amyloliquefaciens* and *B. licheniformis* were also producing functional alpha
amylase (and confirm the one from *B. subtilis*). Detailed protocol can be found in the [**Experiments page**](https://2021.igem.org/Team%3AGroningen/Experiments). We tested different
constructs (see
 [**Parts page**](https://2021.igem.org/Team%3AGroningen/Parts)) and here we provide evidence of the basic working parts (**Figure 11**). The increase in absorbance observed in the graphs is an indication of starch degradation, an indirect measurement
of alpha amylase activity.

SP049 contains Part3 from *A. oryzae*, SP027 has Part3 from *B. subtilis*, Part3 from *B. amyloliquefaciens* and *licheniformis* can be found in SP041 and SP056, respectively. More detailed information
about the composition of each sample can be found on the [**Parts page**](https://2021.igem.org/Team%3AGroningen/Parts).

![Alpha-amylase activity of the different Parts 3, from B. subtilis (BS), A. oryzae (AO), B. amyloliquefaciens (BA) and B. licheniformis (BL).](https://static.igem.org/mediawiki/2021/0/0a/T--Groningen--Figure9.png)

***Figure 11.** Alpha-amylase activity of the different Parts 3, from B. subtilis (BS), A. oryzae (AO), B. amyloliquefaciens (BA) and B. licheniformis (BL). Measurements were taken in triplicates. All the samples show an increase of absorbance (indirect measurement of the activity) in comparison to the baseline.*

### Part 3b

Some of the samples that contained different Parts 3b were also tested in the halo essay. Parts 3b alpha-amylases from *B. amyloliquefaciens*, *B. licheniformis* and *B. subtilis* were evaluated and showed
results, which can be seen in **Figure 12** and **13**. SP001 contains pPGK1, αMF as a secretion signal, **Part 3b from** ***B. subtilis*** and tTDH1 (expressed
in *S. cerevisiae*). SP021 is composed of pPGK1, αMFΔ, **Part 3b from** ***B. amyloliquefaciens*** and tTDH1 (in *S. cerevisiae*). Finally, SP051 has pRNR1, αMF\_no\_EAEA,
**Part 3b from** ***B. licheniformis*** and tTDH1.

In sample SP001 almost no halo was observed when plating the cells. However, the halo becomes clear when the supernatant or the cell lysate is used, showing that alpha-amylase is active both intra and extracellularly. In the case
of sample SP021, a halo was already observed in the cells plated (**Figure 12a**), as well as the supernatant and bead beated cells. The pattern observed in SP051 differs a bit, as alpha-amylase is only active
when lysing the cells, most likely what is not working in this case is the secretion signal, but alpha-amylase is being produced.

![Triplicates of halo test in samples SP001 (red) and SP021 (blue).](https://static.igem.org/mediawiki/2021/4/44/T--Groningen--Figure10.png)

***Figure 12.** Triplicates of halo test in samples SP001 (red) and SP021 (blue). Different products of the cells were used to compare the activity, **a)** Yeast cells, **b)** cells’ supernatant from liquid cultures and **c)** lysate from cells by bead beating them during 10s.*

![Triplicates of halo test in sample SP051 (Part 3b from B. licheniformis).](https://static.igem.org/mediawiki/2021/1/14/T--Groningen--Figure11.png)

***Figure 13.** Triplicates of halo test in sample SP051 (Part 3b from B. licheniformis). Different products of the cells were used to compare the activity, **a)** Yeast cells, **b)** cells’ supernatant from liquid cultures, **c)** lysate from cells by bead beating them during 10s and **d)** lysate from cells by bead beating them during 10s + 30s.*

Some samples were also tested with the alpha-amylase assay kit as explained above. Part 3b from *B. subtilis* and *B. licheniformis* are in SP001 and SP063, respectively. SP002 holds Part 3b from *A. oryzae* and SP031 contains Part 3b from *B. amyloliquefaciens*. Performance of the parts can be seen in **Figure 14**. Some samples were more active than others but all show some activity.

![Triplicates of halo test in sample SP051 (Part 3b from B. licheniformis).](https://static.igem.org/mediawiki/2021/5/5e/T--Groningen--Figure12.png)

***Figure 14.** Alpha amylase activity of the different Parts 3b, from B. subtilis (BS), B. licheniformis (BL), A. oryzae (AO), and B. amyloliquefaciens (BA). Measurements were taken in triplicates. All the samples show an increase of absorbance (indirect measurement of the activity) in comparison to the baseline.*

## Combinatorial library - Subset

Having proved that *Saccharomyces spp.* is capable of producing alpha-amylase, we wanted to go further and see how **the yield** could be improved. The modularity of the Golden Gate Assembly inspired us to try different promoters
(with varied strengths), secretion signals, alpha-amylase genes and yeast strains. The different elements in each category can be found in **Table 1.**

The spectrum of combinations that we could attempt was big, that would have required a lot of DBTL cycles. In order to avoid this, a **set of samples** were selected (**Table 2.**), from all the possible combinations,
those samples were tried experimentally. The knowledge acquired by testing those constructs was used to explore what would be the best combinations through **AI technology**. The total number of possible combinations
were **640**, the different elements are illustrated in **Figure 15**.

***Table 1.** Elements that can be combined to produce alpha amylase.*

![Elements that can be combined to produce alpha amylase.](https://static.igem.org/mediawiki/2021/c/c4/T--Groningen--TableSofia.png)

![Different elements from the categories.](https://static.igem.org/mediawiki/2021/a/a5/T--Groningen--Elements.png)

***Figure 15.** Different elements from the categories. Promoters are represented in green, alpha amylase and secretion signals are yellow and the terminator is red.*

***Table 2.** Subset of 10% of the possible combinations. SP - Signal peptide.*

| Sample | Strain | Promoter | SP | Gene |
| --- | --- | --- | --- | --- |
| SP001 | ySB77 | pPGK1 | αMF | *B. subtilis* |
| SP002 | ySB85 | pRNR1 | αMFΔ | *A. oryzae* |
| SP003 | ySB76 | pTDH3 | αMF | *A. oryzae* |
| SP004 | ySB76 | pRNR1 | αMFΔ | *A. oryzae* |
| SP005 | ySB78 | pHHF1 | αMF | *B. subtilis* |
| SP006 | ySB77 | pRNR1 | αMF\_no\_EAEA | *A. oryzae* |
| SP007 | ySB85 | pHHF1 | αMF\_no\_EAEA | *B. subtilis* |
| SP008 | ySB78 | pHHF1 | Native | *B. subtilis* |
| SP009 | ySB85 | pRNR1 | Native | *B. subtilis* |
| SP010 | ySB77 | pHHF1 | αMF | *B. subtilis* |
| SP011 | ySB77 | pTDH3 | Native | *B. licheniformis* |
| SP012 | ySB78 | pRNR1 | Native | *A. oryzae* |
| SP013 | ySB85 | pRAD27 | αMF | *B. subtilis* |
| SP014 | ySB76 | pREV1 | αMF | *B. amyloliquefaciens* |
| SP015 | ySB76 | pPGK1 | αMFΔ | *A. oryzae* |
| SP016 | ySB77 | pRAD27 | Native | *B. licheniformis* |
| SP017 | ySB85 | pTEF1 | αMFΔ | *A. oryzae* |
| SP018 | ySB76 | pRAD27 | αMFΔ | *B. amyloliquefaciens* |
| SP019 | ySB78 | pTEF1 | Native | *B. licheniformis* |
| SP020 | ySB76 | pRNR1 | αMFΔ | *B. amyloliquefaciens* |
| SP021 | ySB77 | pPAB1 | αMF | *B. subtilis* |
| SP022 | ySB77 | pREV1 | αMF | *B. amyloliquefaciens* |
| SP023 | ySB77 | pRNR2 | αMF\_no\_EAEA | *B. licheniformis* |
| SP024 | ySB77 | pTEF1 | αMFΔ | *B. licheniformis* |
| SP025 | ySB78 | pPAB1 | Native | *B. amyloliquefaciens* |
| SP026 | ySB76 | pHHF1 | αMF | *B. licheniformis* |
| SP027 | ySB76 | pRNR1 | Native | *B. subtilis* |
| SP028 | ySB77 | pTEF1 | αMF | *B. amyloliquefaciens* |
| SP029 | ySB85 | pRPL18B | αMF | *B. licheniformis* |
| SP030 | ySB85 | pTDH3 | αMFΔ | *B. amyloliquefaciens* |
| SP031 | ySB77 | pPGK1 | αMFΔ | *B. amyloliquefaciens* |
| SP032 | ySB78 | pRPL18B | αMF\_no\_EAEA | *B. amyloliquefaciens* |
| SP033 | ySB85 | pREV1 | αMF | *B. subtilis* |
| SP034 | ySB76 | pTEF1 | αMF | *B. licheniformis* |
| SP035 | ySB76 | pPAB1 | αMF\_no\_EAEA | *B. licheniformis* |
| SP036 | ySB76 | pTDH3 | αMF | *B. amyloliquefaciens* |
| SP037 | ySB85 | pRPL18B | αMF | *B. subtilis* |
| SP038 | ySB78 | pRNR2 | αMF | *B. subtilis* |
| SP039 | ySB85 | pTEF1 | αMF | *B. licheniformis* |
| SP040 | ySB85 | pTEF1 | αMF\_no\_EAEA | *B. licheniformis* |
| SP041 | ySB85 | pHHF1 | Native | *B. amyloliquefaciens* |
| SP042 | ySB76 | pRPL18B | αMF | *B. licheniformis* |
| SP043 | ySB85 | pRPL18B | αMF\_no\_EAEA | *A. oryzae* |
| SP044 | ySB77 | pRNR2 | αMFΔ | *B. licheniformis* |
| SP045 | ySB78 | pPAB1 | αMF | *B. licheniformis* |
| SP046 | ySB77 | pTDH3 | αMFΔ | *B. licheniformis* |
| SP047 | ySB77 | pPGK1 | Native | *A. oryzae* |
| SP048 | ySB78 | pRNR2 | αMF\_no\_EAEA | *B. amyloliquefaciens* |
| SP049 | ySB85 | pRAD27 | Native | *A. oryzae* |
| SP050 | ySB77 | pPGK1 | αMF\_no\_EAEA | *B. amyloliquefaciens* |
| SP051 | ySB76 | pRNR1 | αMF\_no\_EAEA | *B. licheniformis* |
| SP052 | ySB77 | pPAB1 | αMF | *B. amyloliquefaciens* |
| SP053 | ySB85 | pHHF1 | Native | *A. oryzae* |
| SP054 | ySB85 | pRPL18B | αMFΔ | *B. subtilis* |
| SP055 | ySB77 | pRPL18B | αMF | *B. licheniformis* |
| SP056 | ySB78 | pPAB1 | Native | *B. licheniformis* |
| SP057 | ySB85 | pHHF1 | αMF\_no\_EAEA | *B. licheniformis* |
| SP058 | ySB78 | pRNR2 | Native | *A. oryzae* |
| SP059 | ySB77 | pHHF1 | αMFΔ | *B. amyloliquefaciens* |
| SP060 | ySB85 | pREV1 | Native | *B. subtilis* |
| SP061 | ySB85 | pREV1 | αMF\_no\_EAEA | *B. amyloliquefaciens* |
| SP062 | ySB85 | pRAD27 | αMF | *B. amyloliquefaciens* |
| SP063 | ySB76 | pRNR2 | αMF | *B. licheniformis* |
| SP064 | ySB85 | pPGK1 | αMFΔ | *A. oryzae* |

## References

1. UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 49:D1 (2021)
2. Lee, M. E., Deloache, W. C., Cervantes, B., & Dueber, J. E. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth. Biol., 4, 975–986.
3. Obst, U., Lu, T. K., & Sieber, V. (2017). A Modular Toolkit for Generating Pichia pastoris Secretion Libraries. ACS Synthetic Biology, 6(6), 1016–1025.
4. Yan, S., Wu, G. (2017) Bottleneck in secretion of α-amylase in Bacillus subtilis . Microb Cell Fact 16, 124 .

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# Team:Groningen/Experiments

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Groningen 2021

* Project
  + [Description](https://2021.igem.org/Team%3AGroningen/Description)
  + [Model](https://2021.igem.org/Team%3AGroningen/Model)
  + [Contribution](https://2021.igem.org/Team%3AGroningen/Contribution)
  + [Implementation](https://2021.igem.org/Team%3AGroningen/Implementation)
  + [Awards](https://2021.igem.org/Team%3AGroningen/Medals_and_Awards)
* Wet lab
  + [Engineering](https://2021.igem.org/Team%3AGroningen/Engineering)
  + [Notebook](https://2021.igem.org/Team%3AGroningen/Notebook)
  + [Experiments](https://2021.igem.org/Team%3AGroningen/Experiments)
  + [Results](https://2021.igem.org/Team%3AGroningen/Results)
  + [Parts](https://2021.igem.org/Team%3AGroningen/Parts)
  + [Safety](https://2021.igem.org/Team%3AGroningen/Safety)
* People
  + [Team](https://2021.igem.org/Team%3AGroningen/Team)
  + [Attributions](https://2021.igem.org/Team%3AGroningen/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AGroningen/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AGroningen/Partnership)
  + [Sponsors](https://2021.igem.org/Team%3AGroningen/Sponsors)
* Outreach
  + [Human
    Practices](https://2021.igem.org/Team%3AGroningen/Human_Practices)
  + [Integrated Human
    Practices](https://2021.igem.org/Team%3AGroningen/Gold_Human_Practices)
  + [Education](https://2021.igem.org/Team%3AGroningen/Communication)

![](https://static.igem.org/mediawiki/2021/5/53/T--Groningen--ExperimentsBanner.png)

# Experiments

## Introduction

This page presents the collection of wet lab protocols that our team used through the busy working summer. The purpose of this page is to present all the steps and reagents needed in the protocols in detail. To see our weekly wet lab
progress, head over to the [**Notebook page**](https://2021.igem.org/Team%3AGroningen/Notebook). To see the final results in detail, please head over to the [**Results page**](https://2021.igem.org/Team%3AGroningen/Results).
To learn more about our engineering approach, head over to the [**Engineering page**](https://2021.igem.org/Team%3AGroningen/Engineering).

#### Competent *E. coli* DH5α cells

In this protocol, you will find the step-by-step guide on how to create competent *E. coli* DH5α cells. Make sure to keep the buffers in the fridge (4°C) before starting the experiment!

##### Reagents

###### TYM medium

+ 20 g/L bacto-tryptone
+ 5 g/L Yeast extract
+ 0.1 M NaCl
+ 10 mM MgSO4

###### TfB I

+ 30 mM KAc
+ 50 mM MnCl2 (sterilize through filter, add later)
+ 0.1 M KCl
+ 10 mM CaCl2
+ 15% glycerol

###### TfB II

+ 10 mM Na-MOPS, pH=7
+ 75 mM CaCl2
+ 10 mM KCl
+ 15% glycerol

##### Protocol

1. Start ON culture in TYM medium (5mL).
2. On the next morning, measure OD600. Calculate how much to take from the ON culture with the formula: OD600 X V = 0.08 X 300mL.
3. Inoculate 300 mL of TYM medium with the ON culture at OD600=0.08 (200 rpm).
4. Grow cells to OD600=0.6 (around 1.5 hours). Take measurements each half hour until the culture reaches the desired OD600.
5. Cool cell cultures on ice (keep cells cold from now on).
6. Transfer culture in 50 mL tubes.
7. Centrifuge 15 min., GSA rotor, 4200 rpm, 4°C.
8. Discard supernatant, repeat steps 6 and 7 until the whole culture is spun down.
9. Resuspend pellet in 60 mL of cold TfB I (on ice).
10. Centrifuge 10 min., GSA rotor, 4200 rpm, 4°C.
11. Resuspend pellet in 12 mL of cold of TfB II (on ice).
12. Aliquot 300 μl in Eppendorf tubes.
13. Freeze in liquid nitrogen (optional).
14. Store at -80.

#### *E. coli* transformation

In this protocol, you can find how to transform *E. coli* cells (how to insert the desired plasmid into *E. coli*) from competent *E. coli* cells aliquots. Approximate time of the experiment: 2h + overnight incubation.

##### Materials

* Ice
* Prewarm heat block at 42ºC
* Selective plates with the corresponding antibiotics
* Competent cells
* Plasmid DNA

##### Protocol

1. Defrost 150 µl aliquot of *E. coli* competent cells per transformation on ice for 15 min. Label one 1.5 mL microcentrifuge tube for each transformation and then pre-chill by placing the tubes on ice.
2. Add 1 μl plasmid DNA (10 pg/μl), incubate 15 min on ice.
3. Heat shock the cells for 1 min at 42°C.
4. Immediately place the cells on ice for 3 min.
5. Add 1mL LB and incubate at 37°C for 1 hour (200rpm).
6. Plate 100 μl on selective plates.
7. Incubate plates ON at 37ºC.

#### *S. cerevisiae* transformation

Protocol for Lithium Acetate Yeast Transformation.

##### Reagents

###### PLATE Mixture

+ 90mL Sterile 45% PEG 4000
+ 10mL 1M LiOAc
+ 1mL 1M Tris-Cl (pH 7.5)
+ 0.2mL 0.5M EDTA

###### Other materials

+ A solution of 10mg/mL of carrier DNA
+ YPD (Yeast Extract Peptone Dextrose) media
+ Selective plates
+ 1-2µg of Plasmid in ~5µL of water
+ Heat block prewarmed at 42ºC

##### Protocol

1. Inoculate 2 mL cultures for each transformation in YPD and grow ON at 30ºC.
2. In an Eppendorf tube, spin down 1mL of cells (5 seconds, 5000 rpm) for each transformation.
3. Decant the supernatant leaving behind around ~50 μl of the liquid more or less. Resuspend gently.
4. Add 2 μl of 10mg/mL carrier DNA, resuspending cells with pipet tip.
5. Add 5 μl of plasmid and vortex.
6. Add 500 μl of PLATE mixture and vortex.
7. Add 20 μl of 1.0M DTT and vortex.
8. Incubate on your benchtop for at least 3 hours (could go up to 24h). Cells will have settled to the bottom of the tube.
9. Heat shock the cells for 10 min at 42ºC.
10. Place the pipet tip directly into the bottom of the tube and withdraw 100 μl of cells in PLATE mixture (you will get ~75% of the cells in this way).
11. Plate cells on selective media and incubate at 30ºC for 2 days.

##### Notes

1. Cells can be transformed directly from a plate by taking a toothpick of cells and resuspending them in PLATE mix. The transformation from a fresh plate is very efficient, but will still work from an old (several months) plate.
2. Factors that improve efficiency: 1. Late log-phase cells yield more transformants than stationary phase cells 2. Heat shock is not necessary but improves efficiency by 5-fold 3. DTT is not necessary but greatly improves efficiency.
   Use more DTT (up to 100 µL) to further increase efficiency Approximate time for the experiment: Overnight culture + 4-5h (3h min for incubating) + overnight incubation (around 2 days).

#### Part plasmid assembly - BsmBI Golden Gate

Here you can find how to introduce a part into a level 0 vector (pYTK001). Part plasmids are assembled via a BsmBI Golden Gate reaction into the part entry vector. The entry vector contains a **ColE1 origin of replication**,
a **chloramphenicol resistance cassette** and a **GFP expression dropout** for green/white screening. The number of DNA inserts can optionally be normalized to equimolar concentrations (∼20 fmol
each) to improve assembly efficiencies.

##### Reagents

* 4 μL of DNA insert (10 ng/μl)
* 1 μL of Plasmid (20 ng/μl)
* 1 μL T4 DNA Ligase buffer
* 0.5 μL T4 Ligase
* 0.5 μL BsmBI (10000 U/mL)
* Water up to the final volume of 10 μL

##### Protocol

| **Step** | **Temperature (C°)** | **Time** |
| --- | --- | --- |
| Digestion | 42 | 2 min |
| Ligation | 16 | 5 min |
| Final digestion | 60 | 10 min |
| Heat inactivation | 80 | 10 min |
| Hold | 4 | ∞ |

#### Cassette vector assembly - BsaI Golden Gate

In this protocol, you can find how to “build” the final cassette plasmid from the part plasmids. After this digestion and ligation, the transformation into *E. coli* is required to acquire high yields of the
plasmid. The cassette plasmid contains a **kanamycin resistance cassette**.

##### Reagents

* 0.5 μL of part 1-8 plasmids
* 0.5 μL of cassette plasmid
* 1 μL T4 DNA Ligase buffer
* 0.5 μL T4 Ligase
* 0.5 μL BsaI (10000 U/mL)
* Water up to the final volume of 10 μL

##### Protocol

| **Step** | **Temperature (C°)** | **Time** |
| --- | --- | --- |
| Digestion | 42 | 2 min |
| Ligation | 16 | 5 min |
| Final digestion | 60 | 10 min |
| Heat inactivation | 80 | 10 min |
| Hold | 4 | ∞ |

#### PCR amplification of G-Blocks

In this protocol you can find how to amplify the G-Blocks using a 2xPhirePol Green Hot Start Mix mastermix.

##### Reagents

| **Reagent** | **Volume (μl)** |
| --- | --- |
| Master mix | 25 |
| Fw primer (10µM) | 2.5 |
| Rv primer (10µM) | 2.5 |
| Template (50ng) | 1 |
| MilliQ | Up to 50 |

##### Protocol

| **Step** | **Temperature (C°)** | **Time** |
| --- | --- | --- |
| Initial denaturation | 98 | 1 min |
| Denaturation | 98 | 10s |
| Annealing | 60 | 10s |
| Extension | 72 | 30s |
| Final extension | 72 | 1 min |
| Hold | 4 | ∞ |

#### Gel Electrophoresis

In this protocol you can find how to perform gel electrophoresis for analysis of PCR results or digestion of plasmids results.

##### Reagents

* Agarose
* TAE buffer (1x)
* Gel electrophoresis set-up

##### Protocol

###### To make 1% agarose gel:

1. 500 ml TEA buffer
2. 5g agarose
3. Melt in the microwave until the mixture is clear

###### To run the gel electrophoresis:

1. Pour the solution into a gel cast and add 5μl of SYBR green(Invitrogen SYBR Safe DNA gel stain) to the solution. Let the gel set with a comb for slots inserted. Note: SYBR green is light sensitive
2. Add 3 μl of ladder (Quick-Load Purple 1kb Plus DNA Ladder, NEB) to one of the slots.
3. Add 1 μl loading dye (Gel Loading Dye Purple (6x), NEB) to the samples.
4. Run the gel at 120V for 30-40 min.
5. Analyse the gel using a GelDoc (Biorad).

#### 50x TAE buffer

In this protocol you can find how to prepare the stock (50x) TAE buffer to run agarose gels.

##### Reagents

| **Chemical** | **MW** | **Concentration** | **Amount** |
| --- | --- | --- | --- |
| Tris Base | 121.1 g/L | 2 M | 242.2 g/L |
| Acetic Acid | 60.5 mL/L | 1 M | 60.5 mL/L |
| EDTA Sodium salt dihydrate | 372.24 g/L | 50 mM | 18.612 g/L |

##### Protocol

1. Prepare a 500 mM EDTA solution using 93.05 grams of EDTA disodium salt (MW=372.24 g/mol). Dissolve in 400 ml deionized water and adjust the pH NaOH. EDTA will not go completely into solution until the pH is adjusted to about
   8.0!
2. A 50X stock solution is prepared by dissolving 242 g Tris base in 500 mL water, adding 57.1 mL glacial acetic acid, and 100 mL of 500 mM EDTA (pH 8.0) solution, and bringing the final volume up to 1 liter.
3. The 50X stock solution will be diluted 49:1 with water to make a 1X working solution. Final concentrations of the 1X solution are 40 mM Tris, 20 mM acetic acid, and 1 mM EDTA.

#### YPD media

In this protocol you will find how to prepare complex media for growing *Saccharomyces spp.*.

##### Reagents

* Peptone
* Yeast extract
* Agar
* Glucose
* Demiwater

##### Protocol

**Y**east **P**eptone **D**extrose media is composed of YEP media and 2% glucose (from 40% stock). YEP is autoclaved separately and supplemented with Glucose after cooling.

Prepare 500 ml YEP media (**Ye**ast **P**eptone):

| **Compound Name** | **Amount g/0.5L** |
| --- | --- |
| Peptone | 10 |
| Yeast extract | 5 |
| Agar (only if making solid media for plates) | 10 |

Mix components, add 475 ml ddH20 and autoclave. Components will dissolve during autoclaving. After YEP has cooled down, add 25 ml 40% glucose.

#### Antibiotics concentrations

While working in the lab, we used different antibiotics for selection of our plasmids in *E. coli*. Here you can find a summary of the stock and working solutions.

##### Antibiotics:

* Ampicillin
  + Stock solution: 100 mg/mL
  + Working solution: 100 µg/mL
  + Dilution factor --> :1000
  + Results in: 1 µl per ml media
* Chloramphenicol
  + Stock solution: or 25 mg/mL
  + Working solution: 25 µg/mL
  + Dilution factor --> :1000 (25mg/mL stock)
  + Results in: 1 µl per ml media
* Kanamycin
  + Stock solution: 50 mg/mL
  + Working solution: 50 µg/mL
  + Dilution factor --> :1000
  + Results in: 1 µl per ml media

#### Selective Media

In this protocol you will find how to prepare selective media for *Saccharomyces spp.*

##### Reagents

* 20x AA
* Sterile water
* 2x SD
* 10x SD
* 40% Glucose
* 4% Agar
* 0.2% Uracil
* 1% Histidine
* 1% Tryptophan
* 1% Leucine

###### Preparation of 20x AA

Combine amino acid powders in a 1L beaker and add 500 ml ddH2O. Stir under light heating until fully dissolved. Sterile filter into a sterile 500 ml Schott bottle (autoclave the empty bottle before). \*heat sensitive

| **Compound** | **Amount g/0.5L** | **Per liter** | **Final concentration** |
| --- | --- | --- | --- |
| Adenine Sulfate | 0.2 | 400 mg/L | 20 mg |
| Arginine HCL | 0.2 | 400 mg/L | 20 mg |
| Aspartic acid\* | 1 | 2000 mg/L | 100 mg |
| Glutamic acid | 1 | 2000 mg/L | 100 mg |
| Isoleucine | 0.3 | 600 mg/L | 30 mg |
| Lysine HCl | 0.3 | 600 mg/L | 30 mg |
| Methionine | 0.2 | 400 mg/L | 20 mg |
| Phenylalanine | 0.5 | 1000 mg/L | 50 mg |
| Serine | 4 | 8000 mg/L | 400 mg |
| Threonine\* | 2 | 2000 mg/L | 200 mg |
| Tyrosine | 0.3 | 600 mg/L | 30 mg |
| Valine | 1.5 | 3000 mg/L | 150 mg |

###### Preparation of 2x SD

SD = synthetic defined media w/o amino acids and carbon source. Add 6.7 g YNB (Yeast Nitrogen Base without amino acids with ammonium sulfate) per 500 ml ddH2O. Autoclave for 20 min at 120°C. Powder will fully dissolve during
autoclaving.

###### Preparation of 10x SD

SD = synthetic defined media w/o amino acids and carbon source. Add 33.5 g YNB (Yeast Nitrogen Base without amino acids with ammonium sulfate) per 500 ml ddH2O. Autoclave for 20 min at 120°C. Powder will fully dissolve during
autoclaving.

###### Preparation of 40% Glucose

40% (w/v) = 40 g per 100ml. 200 g per 500ml.

Note 1: Glucose will not dissolve easily. Heat up the water first and add glucose little by little while stirring.

Note 2: The Glucose powder takes a significant volume of 500 ml. The final volume is 500 ml including the volume of the glucose.

Weight 200 g glucose (hexose, dextrose) into a beaker. Use a second beaker and add 250 ml ddH2O. Heat water slightly under stirring. Add glucose little by little till fully dissolved. Add additional ddH2O to the 500
ml final volume. Sterile filter into a sterile 500 ml Schott Bottle.

###### Preparation of 4% Agar

4% (w/v) = 4g per 100 ml.

Prepare maximal 300 ml per 500 ml Schott bottle. Add 12 g agar to 300 ml ddH2O and autoclave. Agar will dissolve during autoclaving and solidify when cooled down. Either use directly after autoclaving to pour plates
or heat and dissolve on a heat plate before using it.

###### Preparation of 0.2% Uracil

Add 0.4 g to 200 ml ddH2O and autoclave.

###### Preparation of 1% Histidine, 1% Tryptophan and 1% Leucine

Add 2 g to 200 ml ddH2O and autoclave.

##### Preparation of Selective Media without uracil

To make 1 L of Selective Media (SM) without uracil, mix the following components

| **Component** | **V (mL)** |
| --- | --- |
| 10x SD (regular) | 100 |
| 40% Glucose | 50 |
| 20x AA | 50 |
| 1% histidine | 2 |
| 1% tryptophan | 2 |
| 1% leucine | 9 |
| >demi H20 | 789 |

For making culture plates with SM-ura media, 100 ml 4% agar will be added and 100 ml less demi H2O will be used.

#### Starch test on synthetic alpha-amylase

In this protocol, you can find how the breakdown of starch by synthetic alpha-amylase can be tested using the starch plate halo test.

##### Materials

* Starch plates
  + Culture media + 1 % (potato) starch + 1,5 % agar
* Iodine solution
  + 1 g of iodine crystals + 2 g of potassium iodine in 300 mL of distilled water
* Synthetic alpha-amylase
  + 15 mg/L --> 2 mg of alpha-amylase in 133 mL SM-ura media

##### Protocol

###### Preparation of synthetic alpha-amylase dilutions

1. Prepare **7.5 mg/L alpha-amylase sample solution** by mixing 500 µL of 15 mg/L alpha-amylase sample solution and 500 µL of SM-ura media.
2. Prepare **1.5 mg/L alpha-amylase sample solution** by mixing 100 µL of 15 mg/L alpha-amylase sample solution and 900 µL of SM-ura media.
3. Prepare **0.75 mg/L alpha-amylase sample solution** by mixing 500 µL of 1.5 mg/L alpha-amylase sample solution and 500 µL of SM-ura media.
4. Prepare **0.15 mg/L alpha-amylase sample solution** by mixing 10 µL of 15 mg/L alpha-amylase sample solution and 990 µL of SM-ura media.
5. Prepare **0.075 mg/L alpha-amylase sample solution** by mixing 400 µL of 0.15 mg/L alpha-amylase sample solution and 400 µL of SM-ura media.

##### Spotting of samples

1. Make 2 plates by spotting 5 µL of each sample and the stock solution in triplicate on a starch plate.
2. Incubate 1 plate 24h at 30ºC.
3. Incubate the other plate for 20 min at RT.

##### Halo test

1. Test the breakdown of starch by flooding the surface of the plates with a 1:1 iodine solution with MilliQ for 60 seconds. Discard any excess iodine from the plate.
2. Examine the clear zone around the culture spot. Measure the halo/take a picture from a solid point using the Fujifilm LAS-4000.

#### Alpha-amylase starch test on *Saccharomyces spp.*

In this protocol you can find how the breakdown of starch by alpha-amylase can be tested using the starch plate halo test.

##### Materials

* Starch plates
  + Culture media + 1 % (potato) starch + 1,5 % agar
* Iodine solution
  + 1 g of iodine crystals + 2 g of potassium iodine in 300 mL of distilled water
* ON culture of *Saccharomyces* *spp.*

##### Protocol

###### ON cultures

1. Culture 1 colony of each *Saccharomyces spp.* containing the cassette plasmid in 2 mL 2xSM-ura media overnight shaking at 30ºC.

###### Preparation of culture samples

1. Spin down all the samples at 3000 rpm in 1 min.
2. Dilute the samples to OD600 50

###### Spotting samples

1. Spot 5 µL of each sample in triplicate on a starch plate.
2. Incubate plates for 48h at 30ºC.

###### Halo test

1. Test the breakdown of starch by flooding the surface of the plates with an 1:1 iodine. solution with MilliQ for 60 seconds. Discard any excess iodine from the plate.
2. Examine the clear zone around the culture spot. Measure the halo/take a picture from a solid point using the Fujifilm LAS-4000.

#### Digestion with BsmBI

Digestion with BsmBI was performed to assess the engineering success of all the constructs in the library.

##### Reagents

* DNA template (SP001-SP064 + pRS426-GFP)
* 10x Cut Smart Buffer 3.1 (NEB)
* BsmBI
* MilliQ water

##### Protocol

1. Prepare the digestion Eppendorf tubes as indicated in the table.

   | Components | Volume (μl) |
   | --- | --- |
   | 10x Cut Smart buffer 3.1 | 2.5 |
   | BsmBI enzyme | 0.5 |
   | DNA template (1 μg) | 5 |
   | MQ | 17 |
   | Total | 25 |
2. Incubate the digestion for 1h at 37ºC.
3. Inactivate the enzyme by incubating the reaction at 80ºC for 20 minutes.

#### Alpha-amylase assay kit

This protocol explains how to measure the alpha-amylase activity in the supernatant of overnight cultures using the alpha-amylase assay kit.

##### Reagents

* Amylase Assay Buffer (*Sigma-Aldrich*, Amylase Activity Assay Kit)
* Amylase Substrate Mix (*Sigma-Aldrich*, Amylase Activity Assay Kit)
* Amylase Positive Control (*Sigma-Aldrich*, Amylase Activity Assay Kit)
* Nitrophenol Standard (*Sigma-Aldrich*, Amylase Activity Assay Kit)
* α-Amylase (*Sigma-Aldrich*, α-Amylase from *Aspergillus oryzae*)
  + Molecular weight: 49 - 51 kDa
  + Specific enzyme activity (pH 6.0, 25°C): ~30 U/mg
* Milli-Q water
* YPD media
* 96-well plate (3x)
* 200µL tube (48x)

##### Protocol

###### Preparation

1. Allow Amylase Assay Buffer and α-Amylase to warm up to RT.
2. Set the incubator to 25°C.
3. Transfer cultures into 200 µL tubes (homogenize by pipetting).

###### OD measurement

1. Add 190 µL of Milli-Q water to 48 wells.
2. Add 10 µL of one sample per well (homogenize by vortexing for 20 s).

###### Absorbance measurement

1. Add 0, 2, and 10 µL of **Nitrophenol Standard** in duplicates to 6 different wells on a 96 well plate. Add 50, 48, and 40 µL of **Milli-Q water** into the respective 6 wells on the 96 well
   plate, so the total volume of all wells equals 50µL.
2. Spin down the cultures in 200 µL tubes for 1 min at 8000 rpm, keep the supernatant as a sample.
3. Add 30 µL of each sample in triplicate to a well on a 96 well plate. Add 20 µL of **Amylase Assay Buffer** into the respective wells on the 96 well plate, so the total volume of both wells equals 50
   µL.
4. Prepare **Master Reaction Mix** by mixing 7.1 mL (actually needed: 7.65 mL) of **Amylase Assay Buffer** and 7.1 mL (actually needed: 7.65 mL) of **Amylase Substrate Mix**.
5. Add 100 µL of **Master Reaction Mix** to each well.

#### Obtaining alpha-amylase activity from absorbance measurements

##### Introduction

The change in absorbance over time in the supernatant from overnight liquid media cultures was measured to determine the alpha-amylase activity using the alpha-amylase assay kit. In this protocol you can find how to process the
obtained data.

##### Materials

* Absorbance measurements ($\vec{m}\_i$, where $i$ is the sample number)
* Absolute difference between the starting time and time of taking each of the measurements ($\vec{dt}$)
* Average of duplicate blank standard ($\vec{s}\_B$)
* Average of duplicate 4 nmol standard ($\vec{s}\_4$)
* Average of duplicate 20 nmol standard ($\vec{s}\_{20}$)
* (optional) Average of duplicate 8 nmol standard ($\vec{s}\_8$)
* (optional) Average of duplicate 12 nmol standard ($\vec{s}\_{12}$)
* (optional) Average of duplicate 16 nmol standard ($\vec{s}\_{16}$)

##### Protocol

1. Check whether $\vec{s}\_B$, $\vec{s}\_4$(, $\vec{s}\_8$, $\vec{s}\_{12}$, $\vec{s}\_{16}$) and $\vec{s}\_{20}$ remain constant over time. If all standards show some variation, correct $\vec{m}\_i$ for all $i$ by subtracting with $\vec{s}\_B$
2. Exclude all data points where $\vec{m}\_i>\vec{s}\_{20}$
3. Check whether a linear curve can be fitted to $\vec{m}\_i$ such that $\vec{m}\_i \approx a\_i \* \vec{dt} + b\_i$ for all $i$
4. If the linear curve is a good fit, $a\_i$ is the activity of sample $i$

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# Team:Groningen/Results

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Groningen 2021

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![](https://static.igem.org/mediawiki/2021/0/01/T--Groningen--Results_banner.png)

# Results

* [Introduction](#intro)
* [Ammonia as the only nitrogen source](#ammonia)
* [Engineering success](#engineering)
* [Alpha amylase production](#aAmylase)
* [Improving alpha amylase production](#improving)
* [Final discussion](#discussion)
* [References](#ref)

## Introduction

On this page the main findings and interpretations of the results of our project can be found. In our
project we aimed to achieve four different goals. These four proof of concepts are summarized in
**Figure 1**.

![](https://static.igem.org/mediawiki/2021/4/46/T--Groningen--Results_Figure1.png)

Main proof of concepts that we evidenced during our experiments in the wet and dry lab.

## Ammonia as the only nitrogen source

As we plan on feeding our GMO with ammonia extracted from the MOF, an essential part of our project
is that our GMO should be able to grow in the presence of ammonia as its only nitrogen source. Therefore,
we grew 6 different *Saccharomyces* strains under different ammonia concentrations to
see if the presence of ammonia would affect their growth. In the end we were able to identify the
best performing strains. The protocol can be found in the [Experiments page](https://2021.igem.org/Team%3AGroningen/Experiments).

![](https://static.igem.org/mediawiki/2021/8/86/T--Groningen--Results_Figure2_1.png)

Growth under different ammonia concentrations of different wild type yeast
(Saccharomyces cerevisiae and Saccharomyces paradoxus) strains. The strains tested are a. sYB76
(NCYC3597, S. cerevisiae) b. sYB77 (NCYC3566, S. cerevisiae) c. sYB78 (NCYC3598, S. cerevisiae)
d. sYB79 (NCYC3635, S. paradoxus) e. sYB83 (NCYC3647, S. paradoxus) f. sYB85 (NCYC3687, S.
paradoxus) g. Control (no cells)

As can be seen from **Figure 2**, there are 4 strains that outperformed in the
experiment. These strains were chosen for further experimentation. The doubling time of the chosen
strains can be seen in **Table 1**.

Doubling time (TD) for each strain in different ammonia concentrations in
the exponential phase.

| Strain TD (min) | | | |
| --- | --- | --- | --- |
| 0.05 g/L 5 g/L 7.5 g/L | | |
| ySB76 | 51,71 | 70,71 | 69,3 |
| ySB77 | 130,75 | 90 | 60,78 |
| ySB78 | 101,91 | 94,93 | 88,85 |
| ySB85 | 103,43 | 91,18 | 108,28 |

#### Conclusion

We learned that two of the strains that we were planning to use didn’t perform well when
ammonia was the only source of nitrogen. This experiment helped us to exclude them for further
tests as they weren’t suitable for our purpose (see [Description page](https://2021.igem.org/Team%3AGroningen/Description)). Four strains
proved to grow in different concentrations of ammonia, with strain ySB76 (*S.
cerevisiae*) being the fastest grower overall. We could not observe a clear tendency
that would suggest the optimal ammonia concentration for growing the cells. This result is
relevant for our project, since the concentration of this nutrient doesn't necessarily need to
be kept constant, which allows for more flexibility in ammonia concentration of the cell culture
and thus more flexibility in how much ammonia the MOF should be able to capture. As we wanted to
test different chassis in the optimization process, after this experiment we decided to express
alpha amylase in yYS76, yYB77, ySB78 and ySB85, which correspond with 3 *S. cerevisiae*
strains and 1 *S. paradoxus* strain.

## Engineering success

For our second proof of concept, we wanted to demonstrate that it is possible to use Golden Gate
Assembly to clone heterologous genes for alpha-amylase in *Saccharomyces spp*. Going from the
gene of interest to the cassette plasmid expressed in *Saccharomyces spp.* is a long process.
Throughout it there were some checkpoints that we used to confirm the success of the cloning: GFP
screening; DNA concentration measurements with a Nanodrop; basic parts sequencing; and digestion of
the final cassette plasmids.

### GFP screening

The empty plasmid (pRS426\_\_ConLS'-GFPdropout-ConRE'-URA3-2micron-Kan) used in the Golden Gate
cassette plasmid assemblies contains a **GFP dropout.** This way, when the assembled
plasmid is inserted correctly, the GFP insert will dropout and white colonies will be formed.
Whenever the plasmid was not inserted correctly, the GFP dropout will still be present and colonies
will appear green when examined under UV-light (**Figure 3**). With this simple technique
we make sure that the chosen colonies have uptaken the assembled plasmid without the need of
performing sequencing or digestion experiments prior to plasmid isolation. This GFP screening was
used both for assembling our part plasmids and cassette plasmids.

![](https://static.igem.org/mediawiki/2021/2/26/T--Groningen--Results_Figure3.png)

Screening in *E. coli* colonies with the cassette plasmid SP034. The green fluorescent
colony can be found underneath the cross, the rest appears white coloured. Only white colonies
were selected for plasmid isolation.

#### Conclusion

GFP screening smoothened the process, considering the amount of samples that we needed to
assemble. Generally, all the transformed *E.coli* with cassette plasmids (64 plates) showed a high
efficiency of the Golden Gate assembly; a few countable green colonies were observed in contrast
to hundreds of white colonies. One sample (SP007) didn’t show any white colonies, the
sample was excluded for future experiments.

### DNA concentrations

After purification of cassette plasmids amplified in *E. coli*, DNA concentrations were
measured in order to assure a successful purification prior to *Saccharomyces spp.*
transformations, results can be found in **Table 2**. These measurements were performed
using a nanodrop spectrophotometer, measuring the DNA concentration at a spectrum of wavelengths.

DNA concentration and purity after *E. coli* transformation of cassette plasmid and
plasmid isolation.

| Sample number | ng/ul | A260/280 ratio | Sample number | ng/ul | A260/280 ratio |
| --- | --- | --- | --- | --- | --- |
| SP001 | 22,6 | 1,823 | SP034 | 80 | 0,001 |
| SP002 | 15,6 | 1,803 | SP035 | 35,5 | 1,529 |
| SP003 | 29 | 1,598 | SP036 | 15 | 1,523 |
| SP004 | 19,7 | 1,662 | SP037 | 29,4 | 1,699 |
| SP005 | 54,3 | 2,027 | SP038 | 48,5 | 1,960 |
| SP006 | 53,5 | 1,508 | SP039 | 14,6 | 2,021 |
| SP008 | 69,5 | 1,853 | SP040 | 18,3 | 1,649 |
| SP009 | 16,1 | 2,414 | SP041 | 25,1 | 1,668 |
| SP010 | 12,1 | 1,898 | SP042 | 45,1 | 1,488 |
| SP011 | 13,3 | 1,430 | SP043 | 15 | 2,150 |
| SP012 | 58,9 | 1,812 | SP044 | 29,9 | 1,472 |
| SP013 | 19,5 | 2,058 | SP045 | 78,4 | 1,708 |
| SP014 | 29,1 | 1,473 | SP046 | 33 | 1,451 |
| SP015 | 28,3 | 1,461 | SP047 | 62,3 | 1,489 |
| SP016 | 52,3 | 1,458 | SP048 | 97,6 | 0,002 |
| SP017 | 72,1 | 1,553 | SP049 | 13,4 | 1,949 |
| SP018 | 34,7 | 1,627 | SP050 | 119 | 0,002 |
| SP019 | 79,8 | 1,788 | SP051 | 21,2 | 1,612 |
| SP020 | 15,7 | 1,627 | SP052 | 56,5 | 1,471 |
| SP021 | 17,9 | 1,729 | SP053 | 6,3 | 2,117 |
| SP022 | 71 | 1,514 | SP054 | 39,2 | 1,798 |
| SP023 | 13,2 | 1,688 | SP055 | 25,7 | 1,490 |
| SP024 | 10,6 | 2,078 | SP056 | 50,7 | 2,069 |
| SP025 | 82,7 | 0,002 | SP057 | 73,3 | 1,806 |
| SP026 | 9,5 | 1,602 | SP058 | 51,7 | 1,987 |
| SP027 | 47,2 | 1,512 | SP059 | 30,6 | 1,363 |
| SP028 | 64,2 | 1,544 | SP060 | 61,6 | 1,936 |
| SP029 | 10,5 | 2,297 | SP061 | 62,4 | 1,848 |
| SP030 | 16,6 | 1,892 | SP062 | 82,4 | 1,741 |
| SP031 | 51,3 | 1,507 | SP063 | 66 | 1,547 |
| SP032 | 45 | 2,067 | SP064 | 58,8 | 1,718 |
| SP033 | 48,7 | 1,617 |  |  |  |

#### Conclusion

The A260/280 ratio represents the purity of the DNA, an ideal sample would have a value of
approximately 1.8. Due to large variation in DNA concentration and A260/280 ratio, 20 μl of each
cassette plasmid was used in the *Saccharomyces spp.* transformation instead of 5 μl.
Overall, the concentration of DNA in the samples after transformation and plasmid isolation were
good and ensured the continuation with *Saccharomyces spp*. transformations.

### DNA sequencing

To ensure that our ordered G-blocks for part 3 and part 3B we made were correct, we sequenced the
basic parts (4 full alpha amylases and 4 alpha amylases without the native secretion peptide)
assembled in the entry vector (more on this on the [Engineering page](https://2021.igem.org/Team%3AGroningen/Engineering)). pYTK001 (entry
vector) with Part 3 and 3b was sequenced using two primers flanking the gene, the vector is used to
compose the cassettes plasmids in further experiments. All sequencing results are summarized in
**Table 3.**

Sequencing results from all the parts.

| Part | Alpha amylase Gene | Mutations | Comments |
| --- | --- | --- | --- |
| Part 3 | From *A. oryzae* | - | Good quality sequence, 100% gene coverage. |
| From *B. amyloliquefaciens* | c.301C>Tp.T101I | The amino acid is not in the substrate binding site of the protein. 100 % gene coverage. |
| From *B. licheniformis* | c.690G>Tp.D231Y | Mutations in the flanking aspartate have shown a decrease in thermostability [1]. No data about our mutation. 100% gene coverage. |
| From *B. subtilis* | - | Good quality sequence, 100% gene coverage. |
| Part 3b | From *A. oryzae* | - | Good forward sequencing, 63% gene coverage. |
| From *B. amyloliquefaciens* | - | Good forward sequencing, 52% |
| From *B. licheniformis* | - | Good forward sequencing, 66% gene coverage. |
| From *B. subtilis* | - | Good forward sequencing, 50% gene coverage. |

#### Conclusion

Generally, the results were quite successful and no major mutations were observed. However, two
clear missense mutations were identified in alpha-amylases from *B. amyloliquefaciens*
and *B. licheniformis* that may have an effect on the protein functionality. It would
have been nice if we had the time to restart the cloning from the beginning, unfortunately, the
cassette plasmids were already assembled and the time was very limited. In Part 3b full coverage
of the gene couldn’t be achieved, repeating the sequencing was planned but sadly we
didn’t have the time.

All in all, the mutations found will be taken into account for further results of those enzymes.

### Constructs digestion

We used the restriction enzyme BsmBI to check for the correct ligation of the different constructs of
the library. There are two sites for the enzyme BsmBI in the vector employed (pRS246) for our
constructs, the sites are in part 1 and part 5, on both sides of the insert of interest
(**Figure 4**). The use of BsmBI allows us to check for the size (**Table
4**) of the construct and assess the success of the assembly. The results of the
digestion can be found in **Figure 5**.

![](https://static.igem.org/mediawiki/2021/1/17/T--Groningen--Results_Figure4_2.png)

Scheme of one of the constructs from the library, SP001, where the sites for the enzyme
BsmBI are displayed.

Expected sizes of the different constructs of the library. The elements of each
sample can be found in the pages of the Parts plasmids.

| Sample | Expected sizes A (bp) | Expected sizes B (bp) | Sample | Expected sizes A (bp) | Expected sizes B (bp) |
| --- | --- | --- | --- | --- | --- |
| SP001 | 3729 | 3387 | SP034 | 3729 | 2983 |
| SP002 | 3729 | 2869 | SP035 | 3729 | 2970 |
| SP005 | 3729 | 3387 | SP037 | 3729 | 3387 |
| SP006 | 3729 | 2953 | SP038 | 3729 | 3387 |
| SP008 | 3729 | 3237 | SP039 | 3729 | 2982 |
| SP009 | 3729 | 3237 | SP040 | 3729 | 2970 |
| SP010 | 3729 | 3387 | SP041 | 3729 | 2802 |
| SP011 | 3729 | 2776 | SP042 | 3729 | 2982 |
| SP012 | 3729 | 2755 | SP043 | 3729 | 2953 |
| SP013 | 3729 | 3381 | SP044 | 3729 | 2886 |
| SP014 | 3729 | 2982 | SP045 | 3729 | 2982 |
| SP015 | 3729 | 2869 | SP046 | 3729 | 2866 |
| SP016 | 3729 | 2790 | SP047 | 3729 | 2755 |
| SP017 | 3729 | 2869 | SP048 | 3729 | 2970 |
| SP018 | 3729 | 2880 | SP049 | 3729 | 2749 |
| SP019 | 3729 | 2796 | SP050 | 3729 | 2970 |
| SP020 | 3729 | 2886 | SP051 | 3729 | 2970 |
| SP021 | 3729 | 3387 | SP052 | 3729 | 2982 |
| SP022 | 3729 | 2982 | SP053 | 3729 | 2755 |
| SP023 | 3729 | 2970 | SP054 | 3729 | 3291 |
| SP024 | 3729 | 2886 | SP055 | 3729 | 2982 |
| SP025 | 3729 | 2802 | SP056 | 3729 | 2796 |
| SP026 | 3729 | 2982 | SP057 | 3729 | 2970 |
| SP027 | 3729 | 3237 | SP058 | 3729 | 2755 |
| SP028 | 3729 | 2982 | SP059 | 3729 | 2886 |
| SP029 | 3729 | 2982 | SP060 | 3729 | 3237 |
| SP030 | 3729 | 2866 | SP061 | 3729 | 2970 |
| SP031 | 3729 | 2886 | SP062 | 3729 | 2976 |
| SP032 | 3729 | 2970 | SP063 | 3729 | 2982 |
| SP033 | 3729 | 3387 | SP064 | 3729 | 2869 |
| pRS426-GFP | 3729 | 1338 |  |  |  |

![](https://static.igem.org/mediawiki/2021/4/47/T--Groningen--Results_Figure5.png)

Digestion of the library of constructs with the enzyme BsmBI.

#### Conclusion

After the digestion of all the constructs it can be seen that the expected sizes of the
engineered plasmids match the sizes of the bands shown in the gel. The efficiency of the Golden
Gate assembly is high. There are two samples in which a band of the size of the GFP-drop out is
observed, SP012 and SP058, besides the expected size. This result may be due to contamination
while pipetting. Nevertheless, the expected band can be observed, the samples were used in follow
up experiments, consideration on alpha amylase activity will be taken into account.
Additionally, some samples (SP006, SP011, SP022, SP023, SP044, SP046, SP047, SP052, SP053,
SP055) show faint bands, likely evidencing that the concentration of DNA was low in the first
place. The faint bands have the expected size, and considering the high efficiency in the rest
of the digests we conclude that our Golden Gate assemblies and further screening was highly
successful. No samples were excluded after digestion.

## Alpha amylase production

For our third proof of concept, we wanted to show that our GMO does indeed show alpha-amylase
activity after taking up our construct. We aimed to measure the alpha-amylase production of 64
samples listed at our [Engineering
page](https://2021.igem.org/Team%3AGroningen/Engineering#prod). This was tested with two main experiments. The principal one being an alpha amylase
kit assay used to perform **high throughput** experiments and obtain
**quantitative** data, needed for our model (see [Model page](https://2021.igem.org/Team%3AGroningen/Model)). Additionally, a
**qualitative assay** was employed to confirm the production of alpha-amylase in some
of the samples. To understand more how this assay works, see the [Experiments page](https://2021.igem.org/Team%3AGroningen/Experiments).

### Alpha-amylase starch breakdown

Functionality of alpha-amylase can **qualitatively** be proved by observing the breakdown
of starch. The ability to break down starch is only available when amylase is produced, which
in the case of *S. cerevisiae* and *S. paradoxus* doesn’t happen natively
(**Figure 6e**). However, the assembled constructs should be able to break down starch.
In order to confirm this, selective media plates without uracil (specific for the assembled
constructs) were created with the addition of 1% starch. Several samples were cultured ON and
treated before being plated on the starch plates, as described in the [notebook](https://2021.igem.org/Team%3AGroningen/Notebook) (wk 39), after which pictures
were taken (**Figure 6**) of the with/without iodine treated starch plates. Samples
tested with this assay can be found in **Table 5**.

Composition of samples that were tested by the Halo assay.

| Sample | Yeast Strain | Promoter | Gene | Terminator |
| --- | --- | --- | --- | --- |
| SP001 | ySB77 | pPGK1 | αMF + BS | tTDH1 |
| SP010 | ySB77 | pHHF1 | αMF + BS | tTDH1 |
| SP021 | ySB77 | pPGK1 | αMFΔ + BA | tTDH1 |
| SP026 | ySB76 | pHHF1 | αMF + BL | tTDH1 |
| SP027 | ySB76 | pRNR1 | BS | tTDH1 |
| SP034 | ySB76 | pTEF1 | αMF + BL | tTDH1 |
| SP035 | ySB76 | pPAB1 | αMF\_no\_EAEA + BL | tTDH1 |
| SP051 | ySB76 | pRNR1 | αMF\_no\_EAEA + BL | tTDH1 |
| SP063 | ySB76 | pRNR2 | αMF + BL | tTDH1 |

![](https://static.igem.org/mediawiki/2021/4/4c/T--Groningen--Results_Figure6.png)

Halo assay for 9 constructs and non engineered *Saccharomyces spp.* in triplets,
each plate contains 3 samples, SP001, SP010 and SP021 in plate 1 (left one), SP026, SP027, SP034
in plate 2 (middle) and SP035, SP061 and SP063 in plate 3 (right). Cells were pretreated
differently to check for intra- or extracellular alpha amylase activity. A) Pure cells, B)
Supernatant of overnight culture, C) Lysate of cells by bead beating 10s, D) Lysate of cells by
bead beating during 10s + 30s.

#### Conclusion

As can be seen in **Figure 6**, a larger halo was observed in the cells’ lysate samples. Halos were
also visible in most of the supernatant samples. This implies that alpha-amylase is present
**extra- and intracellular** after cell lysis. We were able to confirm that this
activity was only produced in the strains that had taken up the created constructs, as the non
engineered strains didn’t show any halo at all. Overall, we see a difference in the alpha-amylase activity when samples were treated differently, suggesting that the secretion of the
enzyme varies between constructs. More specifically to our project, we planned to isolate the
protein from the supernatant, this way the processing of the cells is minimal and lysating is
not directly necessary. Therefore, it was interesting to find that lysating some of the samples
did produce a bigger halo, since this could change our proposed way of extracting alpha-amylase.
Only few samples of the library were tested through this essay, therefore general conclusions
can not be drawn, but confirmation of alpha amylase-activity could be assessed.

### Alpha-amylase assay kit

We used a colorimetric assay to obtain **quantitative data** of the activity of the
alpha amylase produced by the engineered yeast, allowing for comparison between samples and for
quantitative data to use in our model.

![](https://static.igem.org/mediawiki/2021/8/8c/T--Groningen--Results_Figure7_2.png)

Activity of alpha amylase in a random set of samples from the library. The sample
number refers to the SP0 (being #3 the same as SP003). Activity is measured as the absolute
change in absorbance due to the appearance of p-nitrophenol after 3h.

The assay is based on the capacity of alpha amylase to cleavage ethylidene-pNP-G7, resulting in
**p-nitrophenol**, a product that can be read at a wavelength of 405 nm, more
details on the [Experiments page](https://2021.igem.org/Team%3AGroningen/Experiments).
A testing round was first performed (**Figure 7**) in duplicates per sample to
check how the experiment was done and identify possible steps that may need to be optimized.
This round also helped to exclude samples SP003, SP004 and SP036 for further analysis as we were
tight on reagents and the results weren’t promising.

During the high throughput experiments, we tested the capacity to produce **functional
alpha-amylase** of all the samples which were not excluded from the subset of the
combinatorial library (the excluded samples are listed on the [Model page](https://2021.igem.org/Team%3AGroningen/Model#art1), section Accounting for
lost samples). Activity of alpha-amylase in triplicates of each sample can be found in
**Figure 8**. Supernatants from overnight cultures were incubated with
ethylidene-pNP-G7 in 96 well plates and the appearance of p-nitrophenol was measured every 5 min
during the first hour, then once after 2h and 3h. If alpha-amylase was in the supernatant, an
**increase of absorbance** was expected due to the appearance of p-nitrophenol, an
indirect measurement of alpha-amylase activity. Therefore, activity (y-axis of **Figure 8**)
was measured as the increase of absorbance (quantity of p-nitrophenol) that appears
per time unit (d[p-nitrophenol]/dt).

![](https://static.igem.org/mediawiki/2021/b/b5/T--Groningen--Results_Figure8.png)

High throughput experiment on alpha amylase activity. We tested the activity of
alpha amylase in triplicates in 60 samples. The different graphs account for 3 different
96-well plates a) Samples measured together in a 96-well plate on October 2nd, b) Samples
measured together in a 96-well plate on October 5th and c) Samples measured together in a
96-well plate on October 5th.

After having excluded the samples in which the variance between replicates deviated too much from
the average variance, a table summarizing the mean activity of each sample can be found below.
The different combinations from the subset library are also displayed in **Table
6**.

Alpha-amylase activity of the construct from the subset of combinations. The
different components from each sample are also displayed.

| Sample | Strain | Promoter | Secretion signal | Gene | Activity values (d[p-nitrophenol]/dt) |
| --- | --- | --- | --- | --- | --- |
| SP001 | ySB77 | pPGK1 | αMF | *B.subtilis* | 0,005476099 |
| SP002 | ySB85 | pRNR1 | αMFΔ | *A.oryzae* | 3,4293E-05 |
| SP005 | ySB78 | pHHF1 | αMF | *B.subtilis* | 0,000260468 |
| SP008 | ySB78 | pHHF1 | Native | *B.subtilis* | 0,000192798 |
| SP009 | ySB85 | pRNR1 | Native | *B.subtilis* | 0,00052307 |
| SP010 | ySB77 | pHHF1 | αMF | *B.subtilis* | 0,001100158 |
| SP012 | ySB78 | pRNR1 | Native | *A.oryzae* | 9,26803E-05 |
| SP013 | ySB85 | pRAD27 | αMF | *B.subtilis* | 0,000945715 |
| SP014 | ySB76 | pREV1 | αMF | *B.amyloliquefaciens* | 0,00001 |
| SP015 | ySB76 | pPGK1 | αMFΔ | *A.oryzae* | 1,44142E-05 |
| SP016 | ySB77 | pRAD27 | Native | *B.licheniformis* | 1,2205E-05 |
| SP018 | ySB76 | pRAD27 | αMFΔ | *B.amyloliquefaciens* | 0,00001 |
| SP019 | ySB78 | pTEF1 | Native | *B.licheniformis* | 1,08206E-05 |
| SP022 | ySB77 | pREV1 | αMF | *B.amyloliquefaciens* | 1,14565E-05 |
| SP023 | ySB77 | pRNR2 | αMF\_no\_EAEA | *B.licheniformis* | 0,00001 |
| SP024 | ySB77 | pTEF1 | αMFΔ | *B.licheniformis* | 0,000341834 |
| SP025 | ySB78 | pPAB1 | Native | *B.amyloliquefaciens* | 1,15319E-05 |
| SP026 | ySB76 | pHHF1 | αMF | *B.licheniformis* | 0,000200209 |
| SP027 | ySB76 | pRNR1 | Native | *B.subtilis* | 0,001931731 |
| SP028 | ySB77 | pTEF1 | αMF | *B.amyloliquefaciens* | 1,20322E-05 |
| SP029 | ySB85 | pRPL18B | αMF | *B.licheniformis* | 5,32401E-05 |
| SP030 | ySB85 | pTDH3 | αMFΔ | *B.amyloliquefaciens* | 0,00001 |
| SP031 | ySB77 | pPGK1 | αMFΔ | *B.amyloliquefaciens* | 2,44344E-05 |
| SP032 | ySB78 | pRPL18B | αMF\_no\_EAEA | *B.amyloliquefaciens* | 1,85894E-05 |
| SP033 | ySB85 | pREV1 | αMF | *B.subtilis* | 0,000845811 |
| SP034 | ySB76 | pTEF1 | αMF | *B.licheniformis* | 0,000211464 |
| SP035 | ySB76 | pPAB1 | αMF\_no\_EAEA | *B.licheniformis* | 0,00021031 |
| SP037 | ySB85 | pRPL18B | αMF | *B.subtilis* | 0,00113086 |
| SP038 | ySB78 | pRNR2 | αMF | *B.subtilis* | 0,002933777 |
| SP039 | ySB85 | pTEF1 | αMF | *B.licheniformis* | 1,41915E-05 |
| SP041 | ySB85 | pHHF1 | Native | *B.amyloliquefaciens* | 1,17257E-05 |
| SP044 | ySB77 | pRNR2 | αMFΔ | *B.licheniformis* | 4,16163E-05 |
| SP045 | ySB78 | pPAB1 | αMF | *B.licheniformis* | 9,44327E-05 |
| SP046 | ySB77 | pTDH3 | αMFΔ | *B.licheniformis* | 1,15677E-05 |
| SP047 | ySB77 | pPGK1 | Native | *A.oryzae* | 0,00019171 |
| SP049 | ySB85 | pRAD27 | Native | *A.oryzae* | 0,00029359 |
| SP050 | ySB77 | pPGK1 | αMF\_no\_EAEA | *B.amyloliquefaciens* | 1,39692E-05 |
| SP051 | ySB76 | pRNR1 | αMF\_no\_EAEA | *B.licheniformis* | 0,000290985 |
| SP052 | ySB77 | pPAB1 | αMF | *B.amyloliquefaciens* | 1,01931E-05 |
| SP053 | ySB85 | pHHF1 | Native | *A.oryzae* | 1,35179E-05 |
| SP054 | ySB85 | pRPL18B | αMFΔ | *B.subtilis* | 0,000154906 |
| SP055 | ySB77 | pRPL18B | αMF | *B.licheniformis* | 0,00001 |
| SP056 | ySB78 | pPAB1 | Native | *B.licheniformis* | 1,94392E-05 |
| SP058 | ySB78 | pRNR2 | Native | *A.oryzae* | 6,79755E-05 |
| SP060 | ySB85 | pREV1 | Native | *B.subtilis* | 0,000223757 |
| SP061 | ySB85 | pREV1 | αMF\_no\_EAEA | *B.amyloliquefaciens* | 1,40926E-05 |
| SP062 | ySB85 | pRAD27 | αMF | *B.amyloliquefaciens* | 1,38021E-05 |
| SP063 | ySB76 | pRNR2 | αMF | *B.licheniformis* | 0,000596797 |

#### Conclusion

Experiment were performed in triplicate to account for stochasticity, samples SP006, SP011,
SP020, SP021, SP042 and SP059 were excluded, as the variance between replicates deviated from
the mean variance, evidencing that the signal was more likely an artefact than actual alpha
amylase activity. For the rest of the samples displayed in the graphs it can be concluded that
there is a high variability in terms of how active alpha amylase is. Some constructs
aren’t successful in expressing active alpha amylase in the supernatant (any sample with
an activity value below 0,00001 was considered non active). However, other samples produce
bigger amounts of p-nitrophenol and thus have more alpha-amylase activity in the supernatant.

Some **general trends** can be observed by looking at the graphs and the table, for
instance, a relatively high alpha amylase activity is observed in samples that contain the alpha
amylase gene from the **host** ***B. subtilis***. The
interpretation of such a big set of samples and identification of the best performers is an
arduous task. AI technology could help getting deeper insights on our library and finding a
better combination from the combinatorial library, more on this can be found in the [model page](https://2021.igem.org/Team%3AGroningen/Model).

## Improving alpha amylase production

We used the Automated Recommendation Tool (ART) to model which samples from the combinatorial library
discussed on the [engineering page](https://2021.igem.org/Team%3AGroningen/Engineering)
would yield a high alpha-amylase activity in the supernatant (exploitative recommendations).
Furthermore, the ART also provides recommendations on which samples should be tested in a future
DBTL-cycle in order to improve the performance of the model (exploratory recommendations). On this
page, only the final results of the ART are discussed. How these results are generated and how to
analyze them is discussed on the [modeling
page](https://2021.igem.org/Team%3AGroningen/Model#art).

### Exploitative recommendations

Exploitative recommendation given by the ART.

| Rank | strain | promoter | secretion | gene | Predicted ln(activity) | Predicted activity |
| --- | --- | --- | --- | --- | --- | --- |
| 0 | ySB78 | pPGK1 | αMF | *B.subtilis* | -6.241298 | 0.001947 |
| 1 | ySB78 | pTDH3 | αMF | *B.subtilis* | -6.323765 | 0.001793 |
| 2 | ySB76 | pRNR2 | αMF | *B.subtilis* | -6.363736 | 0.001723 |
| 3 | ySB85 | pPGK1 | αMF | *B.subtilis* | -6.388392 | 0.001681 |
| 4 | ySB76 | pPGK1 | αMF | *B.subtilis* | -6.435489 | 0.001604 |
| 5 | ySB77 | pRNR2 | αMF | *B.subtilis* | -6.521911 | 0.001471 |
| 6 | ySB77 | pTDH3 | αMF | *B.subtilis* | -6.566358 | 0.001407 |
| 7 | ySB76 | pHHF1 | αMF | *B.subtilis* | -6.573551 | 0.001397 |
| 8 | ySB76 | pRNR1 | αMF | *B.subtilis* | -6.666158 | 0.001273 |
| 9 | ySB76 | pTDH3 | αMF | *B.subtilis* | -6.676021 | 0.001261 |
| 10 | ySB85 | pRNR1 | αMF | *B.subtilis* | -6.689628 | 0.001244 |
| 11 | ySB77 | pRNR1 | αMF | *B.subtilis* | -6.707219 | 0.001222 |
| 12 | ySB76 | pPAB1 | αMF | *B.subtilis* | -6.75746 | 0.001162 |
| 13 | ySB77 | pREV1 | αMF | *B.subtilis* | -6.892306 | 0.001016 |
| 14 | ySB78 | pRNR1 | αMF | *B.subtilis* | -6.892674 | 0.001015 |
| 15 | ySB85 | pPAB1 | αMF | *B.subtilis* | -6.914319 | 0.000993 |
| 16 | ySB76 | pTEF1 | αMF | *B.subtilis* | -6.930234 | 0.000978 |
| 17 | ySB78 | pPAB1 | αMF | *B.subtilis* | -6.938977 | 0.000969 |
| 18 | ySB76 | pREV1 | αMF | *B.subtilis* | -6.942261 | 0.000966 |
| 19 | ySB85 | pTEF1 | αMF | *B.subtilis* | -6.95399 | 0.000955 |
| 20 | ySB77 | pPAB1 | αMF | *B.subtilis* | -6.968985 | 0.000941 |
| 21 | ySB78 | pRPL18B | αMF | *B.subtilis* | -6.980546 | 0.00093 |
| 22 | ySB85 | pTDH3 | αMF | *B.subtilis* | -6.996441 | 0.000915 |
| 23 | ySB77 | pTEF1 | αMF | *B.subtilis* | -7.012753 | 0.0009 |
| 24 | ySB78 | pREV1 | αMF | *B.subtilis* | -7.057184 | 0.000861 |
| 25 | ySB78 | pRAD27 | αMF | *B.subtilis* | -7.06477 | 0.000855 |
| 26 | ySB85 | pRNR2 | αMF | *B.subtilis* | -7.082414 | 0.00084 |
| 27 | ySB78 | pRNR1 | αMF\_no\_EAEA | *B.subtilis* | -7.096215 | 0.000828 |
| 28 | ySB78 | pTEF1 | αMF | *B.subtilis* | -7.134024 | 0.000798 |
| 29 | ySB76 | pRAD27 | αMF | *B.subtilis* | -7.223168 | 0.000729 |

**Table 7** shows that all exploitative recommendations contain the alpha-amylase
encoding genes from *B.subtilis*. Furthermore, the αMF secretion peptide occurs in 29
out of 30 recommendations. It is also striking that 8 out of 10 recommendations contain either the
pRNR2, pTDH3 or pPGK1 promoter, with the pPGK1 promoter occurring in 3 out of the top 5
recommendations. No clear pattern can be noticed in the recommendations regarding which
*Saccharomyces spp.* should be used as a chassis.

### Exploratory recommendations

Exploratory recommendation given by the ART

| Rank | strain | promoter | secretion | gene | Predicted ln(activity) | Predicted activity |
| --- | --- | --- | --- | --- | --- | --- |
| **0** | ySB78 | pTDH3 | αMF | B.amyloliquefaciens | -10.158696 | 0.000039 |
| **1** | ySB78 | pTDH3 | Native | B.amyloliquefaciens | -10.167739 | 0.000038 |
| **2** | ySB78 | pRPL18B | αMF\_no\_EAEA | A.oryzae | -9.694006 | 0.000062 |
| **3** | ySB78 | pPAB1 | αMF\_no\_EAEA | B.licheniformis | -9.807968 | 0.000055 |
| **4** | ySB78 | pTDH3 | αMF | B.subtilis | -6.323765 | 0.001793 |
| **5** | ySB78 | pPGK1 | αMF | B.amyloliquefaciens | -10.157919 | 0.000039 |
| **6** | ySB78 | pTDH3 | Native | B.subtilis | -7.246373 | 0.000713 |
| **7** | ySB78 | pRNR1 | αMF\_no\_EAEA | B.amyloliquefaciens | -10.167606 | 0.000038 |
| **8** | ySB78 | pRNR1 | αMF\_no\_EAEA | A.oryzae | -9.742028 | 0.000059 |
| **9** | ySB78 | pHHF1 | αMF\_no\_EAEA | B.amyloliquefaciens | -10.261079 | 0.000035 |
| **10** | ySB78 | pREV1 | αMFΔ | A.oryzae | -9.40958 | 0.000082 |
| **11** | ySB78 | pPGK1 | αMF | A.oryzae | -9.517837 | 0.000074 |
| **12** | ySB77 | pPGK1 | αMF | A.oryzae | -9.564904 | 0.00007 |
| **13** | ySB77 | pPGK1 | αMF | B.licheniformis | -9.353338 | 0.000087 |
| **14** | ySB78 | pRAD27 | αMF\_no\_EAEA | A.oryzae | -9.827786 | 0.000054 |
| **15** | ySB78 | pRNR1 | αMF\_no\_EAEA | B.licheniformis | -9.966545 | 0.000047 |
| **16** | ySB76 | pTDH3 | αMFΔ | B.licheniformis | -9.201508 | 0.000101 |
| **17** | ySB78 | pREV1 | αMF\_no\_EAEA | A.oryzae | -9.898024 | 0.00005 |
| **18** | ySB78 | pHHF1 | αMF\_no\_EAEA | B.subtilis | -7.240897 | 0.000717 |
| **19** | ySB77 | pREV1 | αMF | A.oryzae | -9.653788 | 0.000064 |
| **20** | ySB77 | pTDH3 | αMF | B.subtilis | -6.566358 | 0.001407 |
| **21** | ySB85 | pRAD27 | αMF\_no\_EAEA | A.oryzae | -9.909267 | 0.00005 |
| **22** | ySB77 | pTDH3 | Native | B.subtilis | -7.454713 | 0.000579 |
| **23** | ySB85 | pRAD27 | αMF | A.oryzae | -9.6954 | 0.000062 |
| **24** | ySB78 | pRNR2 | αMF | A.oryzae | -9.705825 | 0.000061 |
| **25** | ySB78 | pHHF1 | αMF\_no\_EAEA | B.licheniformis | -10.126075 | 0.00004 |
| **26** | ySB77 | pHHF1 | αMF | A.oryzae | -9.733338 | 0.000059 |
| **27** | ySB78 | pPGK1 | αMF\_no\_EAEA | B.licheniformis | -10.143406 | 0.000039 |
| **28** | ySB78 | pTDH3 | αMF\_no\_EAEA | B.amyloliquefaciens | -10.471749 | 0.000028 |
| **29** | ySB78 | pPGK1 | αMF | B.licheniformis | -9.498398 | 0.000075 |

The exploratory results of the ART are shown in **Table 8**. While these patterns might
seem mostly random at first sight, there are some striking patterns that reflect the frequency and
variance distributions shown in **figures 7 and 8** discussed in the “Step 1: Creating a
well-balanced subset of the combinatorial library” section on the [modeling page](https://2021.igem.org/Team%3AGroningen/Model#art1). For instance the pTDH3
promoter which was shown to be underrepresented in the training data, is part of 8 out the 30
exploratorium recommendations. If all promoters were equally distributed across the recommendations,
it should have occurred around 3 times. Also the αMF\_no\_EAEA secretion signal occurs more
often (13 out of 30) than expected (around 7.5 out of 30). However, the ySB76 occurs a lot less (1
out of 30) than expected (around 7.5 out of 30) even though figure 8 on the [modeling page](https://2021.igem.org/Team%3AGroningen/Model#art1) shows it contributes
less to the overall variance in the dataset than the other strains.

#### Conclusion

Based on the results from the ART, it is expected that a device containing the alpha-amylase
encoding gene from *B.subtilis* and the αMF secretion signal will yield a high
alpha-amylase activity in the supernatant. Furthermore, the exploratory recommendations show
some correlations with the frequency and variance distributions of the part-variants shown on
the [modeling page](https://2021.igem.org/Team%3AGroningen/Model#art).

## Final discussion, finding the most active alpha amylase and future perspectives

Overall, we were able to successfully clone and functionally express alpha-amylase in
*Saccharomyces spp*. using Golden Gate Assembly. However, the level of alpha-amylase activity
did differ based on the way of extraction (lysising or not) and the combination of construct and
chassis. Some results by themselves may not appear to be very **conclusive**, but
bringing all of them together helped us to get a better insight on how to find the most active alpha-amylase.

When checking the activity of alpha-amylase the samples that holded the native sequence (Parts 3 and
Parts 3b) from *B. amyloliquefaciens* and *licheniformis,* a **general
trend** was observed, the samples didn’t perform very well (in terms of alpha-amylase
activity). Combining these results together with the sequencing results, it turns out that
those two samples had clear **missense mutations** that may affect the performance of
them. Two possible scenarios could explain the lack of activity, 1. the deficient production of the
enzyme or 2. the lack of functionality. Considering the experiments performed it is hard to tell
what holds true in this case. Performing direct measurements like SDS gel electrophoresis to
identify the presence of the protein would have given us more insights into where the problem was.
**Further experiments** would be needed to check for the consequences of the mutations.
Ideally, we would have avoided unknown mutations by an early detection of them, unfortunately, at the
time we found them all the constructs of the library were already assembled and we didn’t have
time to restart again.

Besides the engineering strategy, a very important part when expressing heterologous proteins is
measuring the **successful expression and activity** of the protein. Using different
assays as we did is a safe and insightful manner of testing the performance of your device. Far from
being redundant, we draw **different but complementary conclusions** from both
experiments. The halo test helped us understand the importance of the secretion of the enzyme. On the other hand,
the high throughput experiment enabled for more precise comparison between samples and proved to be
an efficient way of getting quantitative data from your devices.

In our attempt to screen as many combinations as possible to find the one that would achieve the
**highest yield of functional alpha-amylase**, we combined *in vitro* high
throughput experiments with a machine learning tool (ART). By doing so we expanded the search space,
by not being just restricted to the actual combinations that were tested in the lab. When merging
these two elements (experiments and machine learning), we encountered some aspects that needed to be
taken into consideration. In our case, the subset of samples from the combinatorial library that are
going to be evaluated experimentally needed to be thoroughly selected, trying to find an
**even** and **representative** distribution of all the elements inside
each category (as explained in the [Model
page](https://2021.igem.org/Team%3AGroningen/Model#art)). Another important aspect is the selection of a performance measure that is
representative of what would constitute a “good” device. We selected the activity of
alpha-amylase in the supernatant of the machine to be this performance measure as it represents what
we aimed to achieve (an efficient production of alpha-amylase) and initial results showed that it
can be accurately measured.

There are some improvements that need to be made in the experiments pipeline (see section
Improvements in the [Model page](https://2021.igem.org/Team%3AGroningen/Model#art)). In the
next DBTL cycle those changes would need to be implemented in order to get more accurate
results.

In general, we learnt that when expressing a new gene in a chassis, it is rather unpredictable how
the different elements in the device would interact between each other**.** From our
experience, we believe that by using this kind of technology more knowledge about the performance of
your device can be acquired and the optimal results can presumably be obtained in fewer DBTL cycles.

## References

1. Declerck N, Machius M, Wiegand G, Huber R, Gaillardin C. Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase. J Mol Biol. 2000 Aug 25;301(4):1041-57. doi: 10.1006/jmbi.2000.4025. PMID: 10966804.

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* [Human practices](https://2021.igem.org/Team%3AGroningen/Human_Practices)
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* [Education](https://2021.igem.org/Team%3AGroningen/Communication)

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#### We thank our sponsors:

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[![EV Biotech](https://static.igem.org/mediawiki/2021/7/74/T--Groningen--EVBiotech.png)](https://evbio.tech/)
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[![Groningen university Funds (GUF)](https://static.igem.org/mediawiki/2021/3/3e/T--Groningen--guf.png)](https://www.rug.nl/alumni/support-research-and-education/groninger-university-fund/)
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