iGEM 2022 — Team rubochum

### What we did in the lab (summary):

End of July

* Quikchange of our genes (CYC, AQ)
* Restriction of our vector *pJET1.2* in order to ligate each of our genes to the vector (done for each gene separately)
* Restriction was done with the same restriction enzymes for both the genes and vector (*BamHI* and *Xhol*)
* Ligation and transformation to *DH5α E.coli*  competent cells
  + these cells multiply said plasmid for the purpose of not having to start from the beginning in case a mistake is made during later steps

August-September

* Restriction of our genes that were ligated to the vector in order to ligate them to a new vector, which was *pBlueScript II SK (+)*  using the same restriction enzymes as above
  + Transfer was done through restriction of the plasmids that had the pJET1.2 vector and our genes and through Gel Electrophoresis, the desired fragments were cut and purified
    - in this case, our desired fragments were those of our genes
* Ligation and transformation of the genes and *pBluescript*  to  *DH5α E.coli*  competent cells
* After each transformation, the cells were platted out in LB-medium Ampicillin Agar plates. The developing colonies were then picked to produce cultures.
  + from those cultures we obtained our plasmids using a kit provided by Promega
* After each step, we sent our plasmid products for sequencing in order to ensure that we were on the right path and everything was it should be
* Another method used to check our progress was check-PCR, where through amplification and then Gel Electrophoresis, we could determine if our product had the correct size according to which step we were at that point in time

### Mistakes made during lab work:

* Communication problems between the lab members, lead to mistakes during lab work
  + for example, due to miscommunication, the wrong restriction enzymes were used
* many different people were coming to the lab who had different amounts of information about what was happening at the lab
  + this led to there being a lot of probes that were not clear on what they contained
  + for example, a probe that was used did not have the vector as previously thought
  + also led to using an insert that was known to have worked for later steps
* The lab book was also not thoroughly written, leading lab members to not understand what was previously done.
  + time was lost trying to understand it

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| ![](https://static.igem.wiki/teams/4293/wiki/design-cycle.png)   Figure 1: iGEM Design Cycle | **1. Design:**  (Design principles are used to specify a biological system with an intended function, and models are used to help make a design according to these principles) After agreeing on our topic, we faced the challenge of successfully duplicating a highly repetitive gene insert. We split our team into smaller groups to determine which pathways we should and wanted to follow. We decided to use *E.Coli* because of its simplicity and forgiveness for beginners. In addition, there were already successful approaches to thread production, so we didn't have to start from scratch. After researching the appropriate setup, we started with the primer and gene design, adapted for our system. We quickly realised that we were too inexperienced and had to educate ourselves with books and other literary sources. However, despite extensive research, we were not versed enough in the full design cycle and, thankfully, received support from our instructor. |

**2. Build:** (The desired DNA sequence encoding the biological system is constructed and implemented into a chassis, such as a target organism)
First, we adapted our required restriction interfaces (*XhoI* and *BamHI*) into our genes using the Quickchange procedure in order to obtain a later multiplication of the gene insert. Subsequently, we added further specific interfaces (*XmaI* and *BspEI*) to our genome using the appropriate primers. We used the pJET1.2 blunt end vector to save and amplify our construct. Using restriction enzymes, we cut out our insert, purified it, and inserted it into a suitable duplication vector to obtain the same procedure in duplicate with the restriction enzymes *XmAI, ScaI,* and *BspEI* and *ScaI* a later duplication of the matching fragments.

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| ![](https://static.igem.wiki/teams/4293/wiki/bersicht.jpeg)   Figure 2: Schematic representation of the entire laboratory plan |

**3. Testing:**After successful cloning, we used check PCR to check whether our insert was included or not, followed by restriction. Both methods were checked by gel electrophoresis. In addition, we had the opportunity to check our purified plasmid by sequencing at the beginning.
**4. Learn:**
In the course of the experiment, we unfortunately made many small mistakes that we had to find together, but managed to improve. Some of them are listed with the corresponding solutions.We adjusted them accordingly and had to start again from scratch.
Mistakes made during lab work:

* Communication problems between the lab members, led to mistakes during lab work
  + for example, due to miscommunication, the wrong restriction enzymes were used
* many different people were coming to the lab who had different amounts of information about what was happening at the lab
  + this led to there being a lot of probes with unclear contents
  + for example, a probe that was used did not have the vector as previously thought
  + this also led to using an insert that was known to have worked for later steps
* The lab book was also not thoroughly written, leading lab members to not understand what was previously done.
  + time was lost trying to understand it

 Success we had during lab work:

* Every step, despite the mistakes, was successful such as the ligation and transformation of our vectors with our genes
* lab work and documentation in the lab book became more organized and more thorough
* only the same few lab members were at the lab every day
  + fewer to no future mistakes
* understanding of the steps that were required to be done

Troubleshooting:
Our testing phase revealed that we had to adjust the heat shock for  *E. coli* so we reduced the duration from 45 sec -1 min to 35 seconds.

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| ![](https://static.igem.wiki/teams/4293/wiki/e-coli.png) |

Figure 3: Transformed  *E.coli*  plates.  *E.coli* strain *DH5 alpha* was transformed by using the heat shock method (duration 35 sec). Cell growth occurred overnight in a shaking incubator at 37°C in the ampicillin-containing LB medium. On the left the results of the transformation of pJET1.2. can be seen. The results of the transformation of pBluescript II KS (+) can be found on the right.

We adjusted the restriction time to 24h (over night) due to the use of two not perfectly harmonised restriction enzymes and their buffers. We tried out various concentration and buffer choices. Instead of 0.5 yL we used 2 yL enzymes to compensate for the loss of reactivity due to the different buffer systems and tested the compatibility with the buffer solution.

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| ![](https://static.igem.wiki/teams/4293/wiki/gel-2.jpeg) |

Figure 4: Agarose gel electrophoresis result: Electrophoretic separation was performed for 30 min at 120 V in a 1.5% agarose gel. The GenRuler 1kb, DNA ladder (ThermoFisher,Scientific) was used as the size standard, which can be seen in the left lane (M). The sizes of the marker bands are given in bp to the left of the gel image. A mutation was introduced into the gene using the quickchange method, which introduced the XhoI and BamHI interfaces. These were inserted into pJET1.2 Blunt end and propagated in *E.Coli DH5 alpha*. The purified plasmid was gelled at 20 yL each as digested and undigested. The digestion was carried out at 37 °C for 1 hour. There was no successful fragmentation.

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| ![](https://static.igem.wiki/teams/4293/wiki/gel-3-2.jpeg) |

Figure 5: Agarose gel electrophoresis result: Electrophoretic separation was performed for 30 min at 120 V in a 1.5 % agarose gel. The GenRuler 1kb, DNA ladder (ThermoFisher,Scientific) was used as the size standard, which can be seen in the right (M) lane. The sizes of the marker bands are given in bp to the right of the gel images. The image shows a successful restriction with XhoI and BamHI of AQ with the time of 24 hours. Previously, we had problems with Cyc, which is why the Check-PCR product was added to the gel. However, there was no successful restriction of the Cyc sample after 24 hours. We attributed this finding to the change of laboratory staff, which led to incorrect sample labelling.

As the gene insert duplication progressed, we noticed that the cloning vector was not well suited to interact with the *E. coli* strain *dh5 alpha*, as we obtained a low plasmid yield. (Unfortunately, we could not improve this due to the lack of budget). As part of the Human practice meeting with Dr. Hendrik Bargel, we got an insight into, what is recommended to use a bright spectrum of E. coli strains, to determine which strain results in a high yield and is also capable of working with the insert.
We duplicated the gene insert using restriction enzymes, gel electrophoresis and heatshock and checked the PCR/sequencing after each step.

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| ![](https://static.igem.wiki/teams/4293/wiki/engineering.jpg)   Figure 6: Joint seminar about lab coordination held by Hoang |

Our work found inspiration in the research work of the Chair of Biomaterials of Prof. Dr. Thomas Scheibel at the University of Bayreuth and served to deepen the fundamental laboratory work in synthetic biology. We didn't want to infringe on anyone's patent, which is why we didn't feel comfortable uploading our Parts to the registry and instead refer to the work of the chair if interested.
Gensequenz AQ and C16:
Huemmerich D, Helsen CW, Quedzuweit S, Oschmann J, Rudolph R, Scheibel T. Primary structure elements of spider dragline silks and their contribution to protein solubility. Biochemistry. 2004 Oct 26;43(42):13604-12. doi: 10.1021/bi048983q . PMID: 15491167 .
Gensequenz C16 with modifcation:
Kramer, J.P.M., Aigner, T.B., Petzold, J. et al. Recombinant spider silk protein eADF4(C16)-RGD coatings are suitable for cardiac tissue engineering. Sci Rep 10, 8789 (2020).


### Success we had during lab work:

* Every step, despite the mistakes, was successful such as the ligation and transformation of our vectors with our genes
* Lab work and documentation in the lab book became more organized and more thorough
* Only the same few lab members were at the lab every day
  + Fewer to no future mistakes
* A better understanding of the steps that were required to be done