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iGEM_Athens_2021_AdAPTED

Improve a Part

Introduction

A. Improvement of an existing part: Teamwork in action

iGEM has a vast collection of DNA parts with many different functions and applications. iGEM Registry and the community of Synthetic Biology can benefit vastly by having teams working towards the optimization and improvement of parts that have already been added and documented by different teams.

B. Inspiration

The idea for the Improvement of an Existing Part for us came through an observation of a possible obstacle that a group of the final users of our project could possibly face. AdAPTED aims to make the production of two fundamental PCR reagents (dNTPs & DNA polymerase) accessible, easy and affordable. The genetic circuit that has been designed, expresses the desired genes under the regulation of T7 promoter. This promoter was preferred over other options due to its numerous advantages regarding protein overexpression. However, in order for the T7 promoter to work, there is a non-negotiable prerequisite: the T7 RNA polymerase. This enzyme is able to bind to the T7 LacO promoter and initiate transcription. However, this particular enzyme is not produced naturally by the different bacterial strains or other chassis and model organisms and can only be found in the Escherichia phage T7 (Bacteriophage T7). As a result, it would be a great advantage to create a system that can transfer DNA to bacteria and also encode for the T7 RNA polymerase so the T7 promoter could be used in every strain.

C. Proposed Solution

Taking into consideration the above, and realising that this might be an issue that other iGEMers and researchers have dealt with, we came up with a possible solution. So, we decided to improve pSB1C3 by inserting to the backbone a transcriptional unit that expresses T7 RNA polymerase. By using this plasmid, cloning a gene regulated by a T7 promoter would be possible in any Escherichia coli strain without having to worry whether it does or does not express T7 RNA polymerase.

Theoretical Background

A. T7 RNA Polymerase Background information and function

Utilizing the T7 RNA polymerase and T7 promoter system is one of the most optimal choices for overexpression of proteins in bacterial hosts. For starters, an important advantage is the independence of the RNA polymerase from the biology of the host cell. The T7 RNA polymerase is exclusively used by the target gene, controlled by the T7 promoter, resulting in extraordinarily high yields of production of recombinant proteins. T7 RNA polymerase is also a very active enzyme: it synthesizes RNA at a rate greater than E. coli RNA polymerase and it terminates transcription less frequently. Except for the high activity, T7 RNA polymerase is highly selective for the initiation of transcription when bound to the T7 promoter sequence in comparison to other promoter sequences in E. coli. Finally, T7 RNA polymerase is resistant to antibiotics such as rifampicin that inhibit E. coli RNA polymerase, and consequently, the addition of rifampicin to cells that are producing T7 RNA polymerase results in the exclusive expression of genes under the control of a T7 RNA polymerase promoter (Ausubel, 1990).

Except for the Bacteriophage T7 from which T7 RNA polymerase derives, mutant strains that have been genetically engineered to be optimum for recombinant protein overexpression, also produce T7 RNA polymerase. A strain like that is Escherichia coli BL21(DE3) that carries the T7 RNA polymerase (RNAP) gene under the control of the lacUV5 promoter, a stronger version of the native lacZ promoter inducible with IPTG (Heyde and Nørholm, 2021).

B. pSB1C3

Regarding the choice of pSB1C3 as the plasmid backbone that we decided to improve by adding the gene coding for T7 RNA polymerase, there were plenty of reasons that led us there. pSB1C3 is one of the most popular plasmid of iGEM as it is the Registry's standard shipping backbone. pSB1C3 is also a well-known plasmid backbone with a lot of user experience behind it. Some of pSB1C3 advantages are:

All parts are flanked by the BioBrick Prefix and Suffix, and are BioBrick compatible. For quality control, EcoRI and PstI restriction sites are available to confirm the part identity and analyze the gel results easily.
The VF2 and VR primer sites flank the Prefix and Suffix, making possible the amplification and sequencing with the same set of primers.
pSB1C3 includes also the antibiotic resistance of chloramphenicol. Chloramphenicol is a common lab antibiotic and does not degrade as quickly as ampicillin.
It has a high-copy origin of replication which improves plasmid yield during extraction.

Figure 1: Original plasmids design that includes (left) RNR and TSase TUs, for the production of dNTPs and (right) Pfu polymerase TU, for the production of Pfu polymerase

Figure 2: Representation of the improved pSB1C3 plasmid backbone

Design of new improved plasmid VS Registry Part pSB1C3

With the experimental design of the plasmid improvement, we anticipate the expression of the mRFP1 gene, when controlled by a T7 promoter, only in bacterial strains that naturally produce T7 RNA polymerase or that have been transformed with our proposed improved plasmid. In this case, this will happen when pSB1C3-RFP is expressed in BL21 (DE3) which naturally expresses the T7 RNA polymerase . Similarly, when pT7SB1C3-RFP is expressed in DH5a which does not have in its genome the gene for T7 RNA polymerase, but uses the one encoded in the improved plasmid.

Parts from the registry and NCBI used during Improvement of an existing part experiments
Code	Name	Short description/purpose of usage.
pSB1C3	pSB1C3	Plasmid backbone we improved.
BBa_J64997	T7 promoter	The promoter which regulates mRFP1 transcription and functions only when T7 RNA polymerase is present.
BBa_M36801	Lac UV5 promoter	The same promoter that was used to clone T7 RNA pol to BL21(DE3) (Heyde and Nørholm, 2021).
BBa_B0034	RBS	Strong bacterial ribosome binding site (Elowitz and Leibler, 2000).
BBa_K1362461	mRFP1	Protein that was used to assess if our system works.
BBa_K809602	T7 RNA Pol	It was inserted to the backbone pSB1C3 under the regulation of the lac UV5 promoter.
BBa_B0015	double terminator consisting of rrnBT1-T7TE terminator	Used to stop the transcription of the T7 RNA Pol.

For the experiments of the improvement of pSB1C3, our team designed and created in the lab two different plasmids: pSB1C3-RFP, pT7SB1C3-RFP as well as planned three transformations in both BL21(DE3) and DH5a E. coli strains. We also designed a pT7SB1C3 plasmid backbone that was not used in our experiments and it is the improved backbone of pSB1C3.

Transformations conducted for Improvement of an existing part
Short symbol	Escherichia coli strain	Does it naturally expressT7 RNA polymerase?	Plasmid	RFP
1(B)	BL21(DE3)	yes	pSB1C-RFP	red fluorescence
2(Δ)	DH5a	no	pSB1C3-RFP	no fluorescence
3(p+i)	DH5a	no	pT7SB1C3: improved pSB1C3 with T7 RNA polymerase TU and with the insert of RFP	red fluorescence

The improved part that is considered our final goal and we hope will be useful to other teams is pT7SB1C3 and it is a new plasmid backbone that includes the old sequence of pSB1C3 and the transcriptional unit responsible for the expression of T7 RNA polymerase. More specifically, the TU’s parts are the consistent promoter lacUv5, the Elowitz RBS, the gene that codes for T7 RNA polymerase and the double rnBT1-T7 terminator.

To test if our hypothesis is correct we conducted our experiments using a reporter chromoprotein under the regulation of the T7 Promoter. We chose to use mRFP1, as it is a reporter protein well documented and it would indicate if the system works due to its fluorescence . In that way, two plasmids had to be assembled. The pT7SB1C3 was ordered in four linear fragments and the insert of mRFP1 was also designed and ordered as a gene fragment.

The pT7SB1C3 plasmid was ordered in five linear fragments, able to form a circular plasmid when going through a Golden Gate reaction with BsaI-HF. The experiments that followed were the Golden Gate Assembly of the five fragments (IAP 1-950, IAP 951- 2.500, IAP 2.501 - 3.970, IAP 3.971- 4.891, IAP insert) to form pT7SB1C3-RFP and the digestion and ligation of both pSB1C3 and mRFP1. The detailed protocols and measurements can both be found in the lab notebook. Lastly, the BL21 (DE3) and DH5a strains were transformed with the pSB1C3-RFP plasmid, as well as the DH5a strain was transformed with the pT7SB1C3-RFP plasmid.

DNA parts ordered from Twist in the form of gene fragments
# of fragment	Name of the gene fragment	Length(bp)
1	IAP 1-950	1.054
2	IAP 951- 2.500	1.659
3	IAP 2.501 - 3.970	1.583
4	IAP 3.971- 4.891	1.012
5	IAP insert	991

Gene Fragments used for each plasmid assembly
pSB1C3-RFP	fragments: 1,2,3,4,5
pT7SB1C3-RFP	linearized pSB1C3 (found in distribution kit)fragment 5

Methods

The protocols that were used during these experiments were

Golden Gate Assembly
Digestion and Ligation Protocol
Transformation
Miniprep
PCR and DNA cleanup kit
Restriction digest for identification of each plasmid
PCR

All the above can be found in detail in the Protocols Tab, while exact experimental procedures and measurements that took place are described in the Notebook.

Conclusions

These experiments led successfully to the expected results according to the assumptions we made before the start of the experiments. The goal of this experiment is to see if we can make DH5a E. coli cells to express mRFP1 under the control of the T7 promoter with the improved plasmid backbone.
We observe that BL21(DE3) E. coli cells transformed with the plasmid pSB1C3-RFP (originally backbone) all express mRFP1 since it expresses T7 RNA polymerase due to its genome (Figure 3). Additionally, it is shown that even though E. coli DH5a were transformed with the same plasmid, they show no fluorescence since they do not possess the required polymerase (Figure 4.).
On the contrary, when DH5a E. coli cells are transformed with the improved backbone but with the same insert, they have distinct colonies that produce fluorescent colonies, some with approximately the same intensity of the BL21 (D3) strains (Figure 5). Consequently, the plasmid backbone that has been created is able to be cloned with inserts that are regulated by the T7 promoter. Even though in this system it was used to overexpress mRFP1, other alternatives can be used as genes allowing an overexpression of the desired genes in a wide variety of hosts.

Figure 3: BL21(DE3) E. coli strain transformed with pSB1C3-RFP plasmid

Figure 4: DH5a E. coli strain transformed with pSB1C3-RFP plasmid

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