Revision as of 09:55, 3 October 2013

Constructs

An amyE intergrational backbone

To transform B. subtilis with our developed biobricks we have improved the amyE intergrational backbone from Munich's iGEM team 2012 (BBa_K823023) that had no inducible promoter. We've added the HyperSpank IPTG inducible promoter from Rudner's Lab 2004. This promoter is placed right in front of the prefix, so any biobrick can be inserted in this backbone (BBa_K1085014) and can be induced with IPTG.

In figure 1 a close-up of the promoter is shown.

The HyperSpank promoter has a single nucleotide change (G->T) at the -1 position. This increases the expression levels but also causes leaky expression when IPTG is absence[1]. To improve repression, a second lacO operator site has been inserted 71 bp upstream of the first. (David Rudner, Harvard Medical School)

Figure 1: The HyperSpank promoter with in orange the lacO operators, in red the -35, -10, +1 signals and in green the prefix.

Figure 2: BBa_K1085014 with: The amp gene for ampicillin resistance in E. coli; The cat gene for chloramphenicol resistance in B. subtilis; The HyperSpank promoter with its repressor lacI; The amyE up- and downstream fragments for intergration in the amyE locus.

Promoter activity

BBa_K1085014 was constructed with two variants of GFP (one form our group and one from the biobrick system, BBa_E0840). BBa_K1085014-GFPmg and BBa_K1085014-BBa_E0840 were transformed to B. subtilis. Overnight cultivated strains were diluted 1:100 in fresh medium and grown till OD~0.45 and induced with 1mM IPTG. A 1,2 and 3 hour sample was analysed under the microscope, results are shown in figure 3.

Figure 3
A:

B:

C:

Figure 3: In (a) An overlay picture is shown from the phase-contrast and GFP-channel picture. In (b,c) the intensity in of ~40 cells in the GFP-channel, were analyzed from two pictures. The average intensity (AU) from the cells are plotted above with the standard deviation. In (b) a GFP from our group is shown and in (c)a biobricked GFP (BBa_E0840) is shown in a plot. wt=wildtype, T0= before induction, T1,2,3=#h of induction with IPTG.

Bacillus subtilis silk genes collection derived from Argiope aurantia MaSp2

One of the challenges that the project includes is the production of spider silk. In order to achieve this goal iGEM Groningen designed for you what we call “The Bacillus subtilis silk assembly shop”.
The 'shop' idea follows those conceptual steps:

Design your own silk protein
Browse the iGEM Groningen silk subunits collection:

Spider silk subunit	Biobrick id	Construct Features
Spider silk subunit	Biobrick id	RBS	Strep-tag	Stop codon
E1	BBa_K1085000	25px	25px
E1	BBa_K1085001	25px
E2	BBa_K1085002
E2	BBa_K1085003		25px
Tail	BBa_K1085008			25px
Tail	BBa_K1085009		25px	25px
table 1: table description.

Select the subunits you need
Assemble them together

At this point the silk product is ready to be produced (See production backbone) and employed for the desired purpose.

The Groningen team enriched the iGEM database adding several new Bacillus subtilis BioBricks containing spider silk gene subunits. This provides the iGEM community with a new bunch of parts; which, in the future, will help anyone in assembling its own silk protein and customize it with different features.
It is the first time that silk-coding BioBricks suitable for Bacillus subtilis are introduced into the database. Therefore this work marks the starting point for further studies about silk within the Bacillus subtilis chassis inside the iGEM framework.

The advantages shown by our collection are mainly two which can be summarized as follows:

Less cloning steps The iGEM database provides a lot of basic parts (such as RBS and terminator), but often achieving the desired goal turns out to be really tedious and time consuming, since the creation of a working construct needs to go through a lot of steps. iGEM Groningen took into account this problematic aspect while designing the silk collection, therefore all the silk subunits (each of them coding for the same final protein product) have been created in different versions (see Table 1). The aim of such a design enables the end-user to reach quicker the desired silk protein construct without bothering too much about the molecular issues. This kind of approach helps to make synthetic biology and specifically working with silk protein in Bacilus subtilis production more accessible.
Compatibility with BioBrick standards We are aware that the BioBrick assembly standard is the foundation of the iGEM method, allowing all different laboratories to build on each others work. That is why the silk subunits are compatible with the following standard:
- BBF RFC 10
- BBF RFC 12
- BBF RFC 21
- BBF RFC 23
- BBF RFC 25
- BBF RFC 1000

The aim of the molecular genetic design is to create a handy toolbox in order to produce any desired combination of silk-like genes which can be expressed in Bacillus subtilis for several purposes. Everything is designed within the RFC[23] standard framework. This enables the user to join together all the different parts creating in-frame fusion silk-like proteins.

The following parts have been produced, sequenced and are available into the standard BioBrick submission backbone (pSB1C3):

What kind of spider silk gene is it
What are general problems with this gene
Where did we get it from.
Codon optimisation, solved the problems.

Strep-tag

Why this is needed
For what purposes it comes in handy

Signal peptide

Why is it so important.
The use of existing pathway so not lots of trouble
The signal sequences are nice addition to the registry
how the pathway works and how the silk will be secreted

@@ Line 175: / Line 175: @@
 The advantages shown by our collection are mainly two which can be summarized as follows:
 <ol><li><b>Less cloning steps</b>
-The iGEM database provides a lot of basic parts (as RBS and terminator) which can be employed for the development of the desired synthetic machine, however most of the time it turns out to be really hard to reach the desired constructs. This is mainly due to the tedious and time consuming molecular biology protocol. For this reason all the silk subunits, produced by the Groningen team, have been created in different versions. Each version contains the information necessary in order to produce the final protein. The aim of such a design enables the end-user to reach quicker the desired silk protein construct without bothering too much about the molecular issues. This kind of approach helps to make synthetic biology and specifically working with silk protein production more accessible.</li>
+The iGEM database provides a lot of basic parts (such as RBS and terminator), but often achieving the desired goal turns out to be really tedious and time consuming, since the creation of a working construct needs to go through a lot of steps. iGEM Groningen took into account this problematic aspect while designing the silk collection, therefore all the silk subunits (each of them coding for the same final protein product) have been created in different versions (see Table 1). The aim of such a design enables the end-user to reach quicker the desired silk protein construct without bothering too much about the molecular issues. This kind of approach helps to make synthetic biology and specifically working with silk protein in <i>Bacilus subtilis</i> production more accessible.</li>
 <li><b>Compatibility with BioBrick standards</b>
 We are aware that the BioBrick assembly standard is the foundation of the iGEM method, allowing all different laboratories to build on each others work. That is why the silk subunits are compatible with the following standard:

Team:Groningen/Project/Construct

From 2013.igem.org