Status: 500 Content-type: text/html

Software error:

Global symbol "$groups" requires explicit package name at /websites/parts.igem.org/cgi/lib/User.pm line 84.
Global symbol "$groups" requires explicit package name at /websites/parts.igem.org/cgi/lib/User.pm line 84.
Global symbol "$groups" requires explicit package name at /websites/parts.igem.org/cgi/lib/User.pm line 85.
Compilation failed in require at /websites/parts.igem.org/cgi/lib/IconBar.pm line 12.
BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/lib/IconBar.pm line 12.
Compilation failed in require at /websites/parts.igem.org/cgi/lib/BBWeb.pm line 9.
BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/lib/BBWeb.pm line 9.
Compilation failed in require at /websites/parts.igem.org/cgi/lib/Change.pm line 8.
BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/lib/Change.pm line 8.
Compilation failed in require at /websites/parts.igem.org/cgi/lib/Part.pm line 12.
BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/lib/Part.pm line 12.
Compilation failed in require at /websites/parts.igem.org/cgi/partsdb/putout.cgi line 8.
BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/partsdb/putout.cgi line 8.

For help, please send mail to this site's webmaster, giving this error message and the time and date of the error.

Status: 500 Content-type: text/html

Software error:

Global symbol "$groups" requires explicit package name at /websites/parts.igem.org/cgi/lib/User.pm line 84.
Global symbol "$groups" requires explicit package name at /websites/parts.igem.org/cgi/lib/User.pm line 84.
Global symbol "$groups" requires explicit package name at /websites/parts.igem.org/cgi/lib/User.pm line 85.
Compilation failed in require at /websites/parts.igem.org/cgi/lib/IconBar.pm line 12.
BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/lib/IconBar.pm line 12.
Compilation failed in require at /websites/parts.igem.org/cgi/lib/BBWeb.pm line 9.
BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/lib/BBWeb.pm line 9.
Compilation failed in require at /websites/parts.igem.org/cgi/lib/Change.pm line 8.
BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/lib/Change.pm line 8.
Compilation failed in require at /websites/parts.igem.org/cgi/lib/Part.pm line 12.
BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/lib/Part.pm line 12.
Compilation failed in require at /websites/parts.igem.org/cgi/partsdb/putout.cgi line 8.
BEGIN failed--compilation aborted at /websites/parts.igem.org/cgi/partsdb/putout.cgi line 8.

For help, please send mail to this site's webmaster, giving this error message and the time and date of the error.

Design Notes

USYD's 2021 team aimed to generate a naturally transformable (NT) E. coli. We planned to insert the 23 genes responsible for NT from A. baylyi into a strain of E. coli. We designed eight clusters of genes to do this, and used a novel recombineering strategy called Babushka Blocks.

One key element of the Babushka block design is that each gene cluster needed a unique selectable marker. Selectable markers need to be used in each gene cluster to be able to select for colonies that have successfully incorporated them into the E. coli chromosome. There are many selectable markers available, but we needed to also consider that the clusters need to mostly stay under 5kb as well, due the limits set by the company we are ordering from (Twist).

We found that the following configuration was possible:

Table 1. Assigned selectable markers to gene clusters.

Cluster	Selectable Marker Used	Total Cluster Length (bp)
1	TpR	4174
2	AmpR	4274
3	fosC2	4876
4	CmR	5015
5	GmR	4948
6	TcR	3370
7	malS	2958
8	qacE	5128

This selectable marker would be used in Cluster 5.

Where possible, restriction enzymes were removed to minimise off-target effects. Substitute bases were chosen to most closely match the natural codon frequency in bacteria.