From 2012.igem.org

Revision as of 10:01, 26 September 2012

Golden Gate Standard

On this page, we introduce the Golden Gate Standard to the Registry of Standard Biological parts. We explain in detail, how Golden Gate Cloning works and how it can be made compatible with RFC 10 standard. Moreover, we provide step-by-step protocols for using this new standard.

Introduction

Although BioBrick assembly is a powerful tool for the synbio community because it allows standardized, simple construction of complex genetic constructs from basic genetic modules, it is not the best option when it comes to assembling larger numbers of modules in a short period of time. Furthermore, BioBrick assembly leaves scars between assembled parts, which is not optimal for protein fusion constructs. One popular method which overcomes these obstacles is Gibson Cloning. This method uses an exonuclease to produce sticky ends on overlapping PCR products, a polymerase to fill up single stranded gaps after annealing and a ligase to connect the different parts . Figure 1: Schematic overview of Gibson Assembly (Gibson et al. 2009).

Gibson cloning allows for assembling whole constructs in one reaction and has been used by many iGEM teams over the past years. However, this technique is not compatible with parts provided by the Registry, unless parts are PCR-amplified in order to linearize them and to add overlapping sequences. Furthermore, Gibson cloning requires three different enzymes and can be very tricky. We therefore propose another method called Golden Gate Cloning (or ist derivatives MoClo and GoldenBraid ). Golden Gate Cloning (GGC) can be used to assemble many fragments with very high efficiency in one reaction . Importantly, insert fragments can be cut out of amplification vectors (such as iGEM standard vectors) and assembled in one single reaction.

Mechanism

Golden Gate Cloning exploits the ability of type IIs restriction enzymes (such as BsaI, BsmBI or BbsI) to produce 4 bp sticky ends right next to their binding sites, irrespective of the adjacent nucleotide sequence. Importantly, binding sites of type IIs restriction enzymes are not palindromic and therefore are oriented towards the cutting site (indicated by the arrows in figure 2 restriction site). So, if a part is flanked by 4 bp overlaps and two binding sites of a type IIs restriction enzyme, which are oriented towards the centre of the part, digestion will lead to predefined sticky ends at each side of the part. In case multiple parts are designed this way and overlaps at both ends of the parts are chosen carefully, the parts align in a predefined order (figure 3). In case a destination vector is added, that contains type IIs restriction sites pointing in opposite directions, the intermediate piece gets replaced by the assembled parts – magic! After transformation, the antibiotic resistance of the destination vector selects for the right clones.
Golden Gate Cloning is typically performed as an all-in-one-pot reaction. This means that all DNA parts, the type IIs restriction enzyme and a ligase are mixed in a PCR tube and put into a thermocycler. By cycling back and forth 10 to 50 times between 37°C and 20°C, the DNA parts get digested and ligated over and over again. Digested DNA fragments are either religated into their plasmids or get assembled with other parts as described above. Since assembled parts lack restriction sites for the type IIs enzyme, the parts get “trapped” in the desired construct. This is the reason why Golden Gate Cloning assembles DNA fragments with such exceptional efficiency. We successfully used this approach to assemble whole TAL effectors vector from six different parts and cloned it into an expression vector – all in one reaction (see below).

Merging BioBrick Standard and Golden Gate Cloning

As described above, the overlaps flanking a part determine the position oft the particular part within the construct after GGC. We therefore propose the following two strategies for implementing Golden Gate Cloning within the Registry of standard biological parts.

1. In most cases, iGEM teams seek to assemble so called protein generators, which consist of one part of each of the following categories:

- Promoters
- Ribosome binding sites (RBS)
- Protein coding regions
- and terminators

Since the order of these parts in a protein generator is always the same (promoter first, RBS second etc.), we can attribute a particular pair of overlaps to each of these categories and thereby define the order of the corresponding parts. From our experience with GGC, we propose the following overlaps which have shown no mispairing in our experiments:

(Figure 4 overlaps) We believe that new standards should only be introduced to the Registry of Standard Biological Parts if they are compatible with the existing standards (most importantly with RFC 10). Otherwise, the registry would get functionally split up into smaller part libraries and teams using one standard could not collaborate with teams using another. We therefore were very careful with introducing type IIs restriction sites into iGEM backbones. In this context, choosing the optimal relative position of the type IIs binding site to the BioBrick prefix and suffix restriction sites is essential for preserving idempotency of the RFC 10 standard. Idempotency in this context means that assembling BioBricks results in higher order constructs that meet BioBrick standard requirements (i.e. they are flanked by the four standard restriction sites and do not contain any of them). The type IIs restriction site can principally be placed in three different positions:

1. between prefix/suffix restriction sites and the actual part
2. between the two restriction sites of the prefix and suffix (EcoRI and XbaI or SpeI and PstI, respectively)
3. distal from both RFC 10 restriction sites.

As illustrated in figure 5, only placing the type IIs site between the RFC 10 standard restriction sites maintains idempotency of the BioBrick standard. In each of the other cases, constructs assembled by Golden Gate Cloning are not iGEM standard compatible because they contain RFC 10 standard restriction sites. We therefore propose the following Golden Gate Standard:

Prefix: Figure 6 (Standard, xxxx represents the four basepair overlaps and NN represents two random nucleotides) We built 96 BioBricks using this Golden Gate Standard and were able to differentially use RFC 10 or Golden Gate standard

Protocol:

Since most standard iGEM plasmids contain binding sites of the most common two type IIs restriction enzymes (namely BsaI/Eco31I and BsmBI/Esp3I), we propose using BbsI/BpiI. We have tested this enzyme in various reaction conditions with many different reaction additives (such as ATP or DTT). Although ligase buffer worked best with other type IIs restriction enzymes (in those cases, ligase activity probably was the bottleneck), we had best results with G Buffer (Fermantas) plus several additives using BbsI.
For creating new biobricks by PCR amplifying the corresponding DNA sequences, we propose the following primers:

1. For Promoters:

2. For RBS:

3. For ORF:

4. For Terminators:

After purification of the PCR product, you can digest your linearized iGEM vector and your part with EcoRI and PstI using the following Protocol: