Team:Heidelberg/Contribution

The Legend of Cellda

---

COLLAPSE NAVIGATION

---

• People
  • Team
  • Attributions
  • Sponsors
• Project
  • Overview
  • RNA-Protein Interaction
  • PPR & Pumilio Domains
  • DNA:RNA Triple Helices
  • Split Trans Splicing Ribozymes
  • Proposed Implementation
  • Engineering Success
  • Proof of Concept
  • Contribution
  • Achievements
• Parts
  • Overview
  • RBP Collection
• Modeling
  • Overview
  • RNA Operads
  • Modular RBP Molecular Modelling
• Software
  • Overview
  • PRISM
  • CoNCoRDE
  • 3DOC
  • RISE
• Human Practices
  • Overview
  • Integrated Human Practices
  • Expert Interviews
  • Safety
  • Education
  • Collaborations
  >
• Notebook
  • Overview
  • Methods
  • Materials
• For Judges

Contribution

Golden Gate Cloning and the high-temperature ligase

Using the Marburg Collection
in E. coli

Due to the Covid-19 pandemic we unfortunately only had limited time in the lab. Hence, we could not use the iGEM 3AA cloning method and had to think of something different. Luckily, René Inckemann from the German Association for Synthetic Biology (GASB) introduced us to Golden Gate Cloning and the Marburg Collection. Golden Gate Cloning allows the highly modular assembly of plasmid constructs. For more information about the cooperation with René also see our Integrated Human Practice page. René personally forwarded us the plasmids (which were more than a hundred) at a visit to Marburg. This enabled us to directly start working with a multitude of biobricks. We want to share the experiences we made while working in E. coli with these biobricks and give tips to further iGEM teams on how to use them. We also adapted the protocols of the iGEM 2018 Marburg Team on a high-temperature ligase.

Designing of overhangs when synthezising new parts

Figure 1: Overhang Design

The forward and the reverse primers should be designed in a similar way. There first should be a sitting sequence of at least two nucleotides as the restriction enzymes are endonucleases and only cut in the middle of a DNA strand. Next, the BsmBI cut side is inserted, and then the respective Entry Vector overhangs. The cut site of BsmbI is indicated with the red dotted line. Here the Part will be inserted into the entry vector. The specific Mo-Clo overhang follows and then finally the sequence of your part.

More specifics on how to design your overhang can be found when downloading the Marburg Collection. When designing a coding sequence, remember to remove the start and stop codons. They will develop in the cloning process, as they are in the overhang of the RBS to the coding sequence and in the overhang of the coding sequence to the terminator. If you include them in your coding sequence, they would occur twice in the final plasmid construct.

When creating Fusion Proteins, you can use the designed overhangs to concatenate more coding sequences (again see the Marburg Collection). But remember that an alanine will be inserted between both of your sequences. This may be a problem when cloning protein complexes that are not isolated subunits, for example, a split Cas9. In this case, we would advise designing your own overhangs for the connection between your concatenated proteins, in order to ensure scarless bonding. You could also use 3DOC to assess whether the alanine may be problematic in your construct.

On the other hand, when you concatenate two coding sequences which are split into isolated functional units, like a Ribonuclease and an RNA binding protein, you may want to use a peptide linker between the subunits. This helps to avoid steric inhibition of the concatenated subunits.

Figure 2: Overhang Design with a peptide linker

In order to avoid steric inhibition of two subunits, when concatenating two coding sequences which are split into isolated functional subunits, a peptide liker should be added in the overhang design.

Of course, you have to make sure that your genes are in frame and contain no BsmBI or BsaI cutting sides. We highly suggest doing in silico cloning in software like Geneious before ordering your DNA synthesis.

Golden Gate Cloning

Overall we found that the cloning protocols of the iGEM Marburg Team in 2018 worked well. We highly suggest normalizing your educt plasmids to an equal molar amount when preparing your Level 1 and Level 2 cloning. Like that, you can minimize the number of religations and incorrectly assembled product plasmids. Although bacteria, which take up just the resistance plasmid are fluorescent, we found that still a high amount of religations and incorrectly assembled constructs with the resistance occur, which leads to colonies that grow on the agar plates but are indistinguishable from the correctly assembled constructs.

In Figure 3, one can see bacteria transformed with Level 2 cloning plasmids. However, when preparing this Level 2 cloning a mistake was made - a 1:1 molar ratio of the resistance plasmid to other educt plasmids was inserted in the reaction. This led to a lot of bacteria absorbing the resistance gene and growing on the plate. As there are a lot of bacterial colonies, the bacteria did not grow well, making colony picking strenuous. Also, the amount of wrongly assembled constructs containing a resistance skyrocketed. Over 10 of the colonies not expressing a fluorescent reporter were picked. Colony PCR and Sanger Sequencing showed, that none of the cultures expressed the right construct.
The mistake was, that too much of the resistance gene was added to the Level 2 cloning reaction mixture. The resistance gene should always be added in a molar concentration 1/10-fold compared to the other components.

Figure 3: Agar plate of a Level 2 cloning without a 1:10 diluted resistance gene

On the plate, only white colonies that did not express GFP were picked. With these colonies, colony PCRs and Sanger Sequencing were conducted to check whether the cloning worked. On this plate, more than ten colonies were picked.

In Level 0 cloning we also first experienced some difficulties. In the beginning, the amount of Entry Vector we used was 75 ng and way too high. With this amount of Entry Vector, we always got a lot of green colonies without a cut-out GFP cassette. Therefore, we changed the amount of Entry Vector to 7.5 ng. This reduced the number of green colonies and increased the number of white colonies that could be picked, as you can see in Figure 4.
As we could not determine the concentration of our PCR products, we always used 3 µl of the insert for the Level 0 cloning. For more information about cloning, also check out our protocols.

Figure 4: Agar plates of Level 0 clonings with different amounts of Entry Vector

We first used a too high concentration of Entry Vector which resulted in a lot of green colonies. When we reduced the amount of Entry Vector we got less green colonies and more white colonies.

Colony PCRs

The iGEM Marburg Team 2018 developed a Colony PCR protocol for Vibrio natriegens. In this protocol, they lyse the cells before conducting the PCR. We found that a colony PCR protocol without prior lysis was sufficient to gain good results in E. coli.

Something else we realized was, that a lot helps a lot! When colony picking, one should pick as many colonies as possible. During our time in the wetlab, we often had real picking-parties, in which we picked more than 200 colonies on one day. Picking many colonies is especially advisable for bacteria transformed after Level 1 cloning. Here we would recommend picking five to ten colonies. For bacteria transformed after Level 0 cloning, it was normally sufficient to only pick around three colonies per construct. Unfortunately, none of our Level 2 clonings worked. We just did not have enough time in the lab. Still, we would also recommend picking five to ten colonies per construct.

Figure 5: Picking Parties

We recommend picking a lot of colonies. Even though it is a lot of work, one should pick at least five to 10 colonies of bacteria transformed with Level 1 and Level 2 plasmids, and around three colonies of bacteria transformed with Level 0 plasmids.

What about ligases?

In our time in the lab, we worked with two different ligases. On the one hand, we worked with the T4-Ligase of NEB the iGEM Marburg Team 2018 used. But we also worked with the Hi-T4 Ligase from NEB. For this ligase, we had to adapt the cloning protocols of the iGEM Marburg Team 2018.
We used these thermocycler settings instead:

Table 1: Thermocycler settings for Golden Gate Level 0 cloning. For the T4-Ligase the ligation temperature in Step 2 was changed to 16 °C.

Step
Step 1	37 °C, 1 min 30 sec
Step 2	25 °C, 3 min
Repetition of Step 1 and 2	15 x
Step 3	50 °C, 5 min
Step 4	80 °C, 10 min
Step 5	4 °C, until lid is opened

Table 2: Thermocycler settings for Golden Gate Level 1 cloning. For the T4-Ligase the ligation temperature in Step 2 was changed to 16 °C.

Step
Step 1	37 °C, 2 min
Step 2	25 °C, 5 min
Repetition of Step 1 and 2	50 x
Step 3	50 °C, 10 min
Step 4	80 °C, 10 min
Step 5	4 °C, until lid is opened

Note: We were not able to construct a finished Level 2 cloning construct in our limited time in the lab, but would highly suspect the thermocycler conditions stated in Table 3.

Table 3: Thermocycler settings for Golden Gate Level 2 cloning. For the T4-Ligase the ligation temperature in Step 2 was changed to 16 °C.

Step
Step 1	37 °C, 5 min
Step 2	25 °C, 16 min
Repetition of Step 1 and 2	30 x
Step 3	37 °C, 30 min
Step 4	80 °C, 10 min
Step 5	4 °C, until lid is opened

We did not verify this experimentally but think it also may be worth to try cloning with ligases that have a lower activity than the T4 or Hi-T4 Ligases of NEB. In spite of the non-sticky overhangs, we received many constructs like this (Figure 6) in our Sanger Sequencing results.

Figure 6: Sanger Sequencing results with a missing promotor

We received many constructs in our sanger sequencing or in our colony PCRs in which plasmids where assembled although parts, like the promotor in this case, were depleted from the construct, but the ligase still closed the plasmid.

Constructs like these are practically undifferentiable from the correct constructs on the agar plates and in the colony PCR. They do not express a fluorescent reporter (like the undigested resistance) and have a small size, so their lack is not noticeable on the gel after a colony PCR. We theorize, that when using a ligase with lower activity, the occurrence of such constructs would be minimized enhancing the efficiency when picking colonies.

Conclusion

We were lucky to have received the Marburg Collection from René Ickemann and the iGEM Marburg Team 2018 and want to tell them thank you once more. To use this collection really felt like using a library of biobricks. It was great to see, how much one could profit from the work of other iGEM teams. This is a great iGEM spirit.

All in all, we are of the opinion, that our lab work went well. In the short time of fewer than two months in the lab without an introduction, we were able to construct functional Level 1 plasmids and also started constructing Level 2 plasmids. The Golden Gate Cloning was an interesting new method to learn. It should be further investigated, improved, and also implemented on different model organisms.

With our work we showed, that Golden Gate Cloning and the Marburg Collection can be successfully used in E. coli. We adapted the protocols of the iGEM Marburg Team 2018 (especially in the colony PCR) and developed a new protocol for another ligase, the NEB Hi-T4 Ligase. Futur iGEM teams we want to give the following tips:

A lot of thoughts should be put into the primer design.

In Level 0 cloning, only 7.5 ng of the entry vector should be used.

The resistance gene should always be added in a molar concentration 1/10-fold compared to the other components in Level 1 and 2 cloning.

Cells do not have to be lysed for colony PCRs.

Loooooots of colonies should be picked (Yey picking parties!).
To make this information public for all iGEM teams we added an entry in the expirience page of the entry vector of the Marburg collection BBa_K2560001.